Medical device with coating for capturing genetically-altered cells and methods for using same

ABSTRACT

Therapeutic and drug delivery systems are provided in the form of medical devices with coatings for capturing and immobilizing target cells such as circulating progenitor or genetically-altered mammalian cells in vivo. The genetically-altered cells are transfected with genetic material for expressing a marker gene and a therapeutic gene in a constitutively or controlled manner. The marker gene is a cell membrane antigen not found in circulating cells in the blood stream and therapeutic gene encodes a peptide for the treatment of disease, such as, vascular disease and cancer. The coating on the medical device may be a biocompatible matrix comprising at least one type of ligand, such as antibodies, antibody fragments, other peptides and small molecules, which recognize and bind the target cells. The therapeutic and/or drug delivery systems may be provided with a signal source such as activator molecules for stimulating the modified cells to express and secrete the desired marker and therapeutic gene products.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/360,567, filed on Feb. 6, 2003, and U.S. patent applicationSer. No. 09/808,867, filed on Mar. 15, 2001, which claims benefit ofU.S. provisional application No. 60/189,674, filed on Mar. 15, 2000 andU.S. provisional application No. 60/201,789, filed on May 4, 2000.

FIELD OF INVENTION

The invention relates to medical devices for implantation into vesselsor hollowed organs of patients such as coated stents, stent grafts,synthetic vascular grafts, heart valves, catheters and vascularprosthetic filters for treating various diseases. In particular, theinvention relates to medical devices comprising a coating on the surfacethat contacts blood, which coating is engineered to capture cells on thesurface of the device. The captured cells form a monolayer on thesurface of the device and are useful in many therapeutic applications,such as a drug delivery system or in the treatment of vascular disease.For example, the cells binding to the implanted medical device may benative, progenitor endothelial cells from the circulating blood orgenetically modified in vitro to express and secrete molecules orsubstances in vivo having a local or generalized therapeutic effect inthe patient.

BACKGROUND OF INVENTION

Diseases such as atherosclerosis and cancer are two of the leadingcauses of death and disability in the world. Atherosclerosis involvesthe development of fatty plaques on the luminal surface of arteries.These fatty plaques causes narrowing of the cross-sectional area of theartery. Ultimately, blood flow distal to the lesion is reduced causingischemic damage to the tissues supplied by the artery.

Coronary arteries supply the heart with blood. Coronary artherosclerosisor coronary artery disease (CAD) is the most common, serious, chronic,life-threatening illness in the United States, affecting more than 11million persons. The social and economic costs of coronaryatherosclerosis vastly exceed those of most other diseases. Narrowing ofthe coronary artery lumen affects heart muscle resulting first inangina, followed by myocardial infarction and finally death, and morethan three hundred thousand of those patients die before reaching thehospital. (Harrison's Principles of Internal Medicine, 14th Edition,1998).

CAD can be treated using percutaneous translumenal coronary angioplasty(PTCA). More than 400,000 PTCA procedures are performed each year in theUnited States. In PTCA, a balloon catheter is inserted into a peripheralartery and threaded through the arterial system into the blockedcoronary artery. The balloon is then inflated, the artery stretched, andthe obstructing fatty plaque flattened, thereby increasing thecross-sectional flow of blood through the affected artery. The therapy,however, does not usually result in a permanent opening of the affectedcoronary artery. As many as 50% of the patients who are treated by PTCArequire a repeat procedure within six months to correct a re-narrowingof the coronary artery. Medically, this re-narrowing of the artery aftertreatment by PTCA is called restenosis. Acutely, restenosis involvesrecoil and shrinkage of the vessel. Subsequently, recoil and shrinkageof the vessel are followed by proliferation of medial smooth musclecells in response to injury of the artery from PTCA. In part,proliferation of smooth muscle cells is mediated by release of variousinflammatory factors from the injured area including thromboxane A₂,platelet derived growth factor (PDGF) and fibroblast growth factor(FGF). A number of different techniques have been used to overcome theproblem of restenosis, including treatment of patients with variouspharmacological agents or mechanically holding the artery open with astent. (Harrison's Principles of Internal Medicine, 14th Edition, 1998).

Of the various procedures used to overcome restenosis, stents haveproven to be the most effective. Stents are metal scaffolds that arepositioned in the diseased vessel segment to create a normal vessellumen. Placement of the stent in the affected arterial segment preventsrecoil and subsequent closing of the artery. Stents can also preventlocal dissection of the artery along the medial layer of the artery. Bymaintaining a larger lumen than that created using PTCA alone, stentsreduce restenosis by as much as 30%. Despite their success, stents havenot eliminated restenosis entirely. (Suryapranata et al. 1998.Randomized comparison of coronary stenting with balloon angioplasty inselected patients with acute myocardial infarction. Circulation97:2502-2502).

Narrowing of the arteries can occur in vessels other than the coronaryarteries, including the aortoiliac, infrainguinal, distal profundafemoris, distal popliteal, tibial, subclavian and mesenteric arteries.The prevalence of peripheral artery atherosclerosis disease (PAD)depends on the particular anatomic site affected as well as the criteriaused for diagnosis of the occlusion. Traditionally, physicians have usedthe test of intermittent claudication to determine whether PAD ispresent. However, this measure may vastly underestimate the actualincidence of the disease in the population. Rates of PAD appear to varywith age, with an increasing incidence of PAD in older individuals. Datafrom the National Hospital Discharge Survey estimate that every year,55,000 men and 44,000 women had a first-listed diagnosis of chronic PADand 60,000 men and 50,000 women had a first-listed diagnosis of acutePAD. Ninety-one percent of the acute PAD cases involved the lowerextremity. The prevalence of comorbid CAD in patients with PAD canexceed 50%. In addition, there is an increased prevalence ofcerebrovascular disease among patients with PAD.

PAD can be treated using percutaneous translumenal balloon angioplasty(PTA). The use of stents in conjunction with PTA decreases the incidenceof restenosis. However, the post-operative results obtained with medicaldevices such as stents do not match the results obtained using standardoperative revascularization procedures, i.e., those using a venous orprosthetic bypass material. (Principles of Surgery, Schwartz et al.eds., Chapter 20, Arterial Disease, 7th Edition, McGraw-Hill HealthProfessions Division, New York 1999).

Preferably, PAD is treated using bypass procedures where the blockedsection of the artery is bypassed using a graft. (Principles of Surgery,Schwartz et al. eds., Chapter 20, Arterial Disease, 7th Edition,McGraw-Hill Health Professions Division, New York 1999). The graft canconsist of an autologous venous segment such as the saphenous vein or asynthetic graft such as one made of polyester, polytetrafluoroethylene(PTFE), or expanded polytetrafluoroethylene (ePTFE), or other polymericmaterials. The post-operative patency rates depend on a number ofdifferent factors, including the lumenal dimensions of the bypass graft,the type of synthetic material used for the graft and the site ofoutflow. Excessive intimal hyperplasia and thrombosis, however, remainsignificant problems even with the use of bypass grafts. For example,the patency of infrainguinal bypass procedures at 3 years using an ePTFEbypass graft is 54% for a femoral-popliteal bypass and only 12% for afemoral-tibial bypass.

Consequently, there is a significant need to improve the performance ofstents, synthetic bypass grafts, and other chronic blood contactingsurfaces and or devices, in order to further reduce the morbidity andmortality of CAD and PAD.

With stents, the approach has been to coat the stents with variousanti-thrombotic or anti-restenotic agents in order to reduce thrombosisand restenosis. For example, impregnating stents with radioactivematerial appears to inhibit restenosis by inhibiting migration andproliferation of myofibroblasts. (U.S. Pat. Nos. 5,059,166, 5,199,939and 5,302,168). Irradiation of the treated vessel can cause severe edgerestenosis problems for the patient. In addition, irradiation does notpermit uniform treatment of the affected vessel.

Alternatively, stents have also been coated with chemical agents such asheparin, phosphorylcholine, rapamycin, and taxol, all of which appear todecrease thrombosis and/or restenosis. Although heparin andphosphorylcholine appear to markedly reduce thrombosis in animal modelsin the short term, treatment with these agents appears to have nolong-term effect on preventing restenosis. Additionally, heparin caninduce thrombocytopenia, leading to severe thromboembolic complicationssuch as stroke. Therefore, it is not feasible to load stents withsufficient therapeutically effective quantities of either heparin orphosphorylcholine to make treatment of restenosis in this mannerpractical.

Synthetic grafts have been treated in a variety of ways to reducepostoperative restenosis and thrombosis. (Bos et al. 1998.Small-Diameter Vascular Graft Prostheses:Current Status Archives Physio.Biochem. 106:100-1115). For example, composites of polyurethane such asmeshed polycarbonate urethane have been reported to reduce restenosis ascompared with ePTFE grafts. The surface of the graft has also beenmodified using radiofrequency glow discharge to fluorinate thepolyterephthalate graft. Synthetic grafts have also been impregnatedwith biomolecules such as collagen. However, none of these approacheshas significantly reduced the incidence of thrombosis or restenosis overan extended period of time.

The endothelial cell (EC) layer is a crucial component of the normalvascular wall, providing an interface between the bloodstream and thesurrounding tissue of the blood vessel wall. Endothelial cells are alsoinvolved in physiological events including angiogenesis, inflammationand the prevention of thrombosis (Rodgers G M. FASEB J 1988;2:116-123.).In addition to the endothelial cells that compose the vasculature,recent studies have revealed that ECs and endothelial progenitor cells(EPCs) circulate postnatally in the peripheral blood (Asahara T, et al.Science 1997;275:964-7; Yin A H, et al. Blood 1997;90:5002-5012; Shi Q,et al. Blood 1998;92:362-367; Gehling U M, et al. Blood2000;95:3106-3112; Lin Y, et al. J Clin Invest 2000;105:71-77). EPCs arebelieved to migrate to regions of the circulatory system with an injuredendothelial lining, including sites of traumatic and ischemic injury(Takahashi T, et al. Nat Med 1999;5:434-438). In normal adults, theconcentration of EPCs in peripheral blood is 3-10 cells/mm³ (TakahashiT, et al. Nat Med 1999;5:434-438; Kalka C, et al. Ann Thorac Surg.2000;70:829-834). It is now evident that each phase of the vascularresponse to injury is influenced (if not controlled) by the endothelium.It is believed that the rapid re-establishment of a functionalendothelial layer on damaged stented vascular segments may help toprevent these potentially serious complications by providing a barrierto circulating cytokines, preventing the adverse effects of a thrombus,and by their ability to produce substances that passivate the underlyingsmooth muscle cell layer. (Van Belle et al. 1997. StentEndothelialization. Circulation 95:438-448; Bos et al. 1998.Small-Diameter Vascular Graft Prostheses:Current Status Archives Physio.Biochem. 106:100-115).

Endothelial cells have been encouraged to grow on the surface of stentsby local delivery of vascular endothelial growth factor (VEGF), anendothelial cell mitogen, after implantation of the stent (Van Belle etal. 1997. Stent Endothelialization. Circulation 95:438-448.). While theapplication of a recombinant protein growth factor VEGF in salinesolution at the site of injury induces desirable effects, the VEGF isdelivered after stent implantation using a channel balloon catheter.This technique is not desirable since it has demonstrated that theefficiency of a single dose delivery is low and produces inconsistentresults. Therefore, this procedure cannot be reproduced accurately everytime.

Synthetic grafts have also been seeded with endothelial cells, but theclinical results with endothelial seeding have been generally poor,i.e., low post-operative patency rates (Lio et al. 1998. New conceptsand Materials in Microvascular Grafting: Prosthetic Graft EndothelialCell Seeding and Gene Therapy. Microsurgery 18:263-256) due most likelyto the fact the cells did not adhere properly to the graft and/or losttheir EC function due to ex-vivo manipulation.

Endothelial cell growth factors and environmental conditions in situ aretherefore essential in modulating endothelial cell adherence, growth anddifferentiation at the site of blood vessel injury. Accordingly, withrespect to restenosis and other blood vessel diseases, there is a needfor the development of new methods and compositions for coating medicaldevices, including stents and synthetic grafts, which would promote andaccelerate the formation of a functional endothelium on the surface ofimplanted devices so that a confluent EC monolayer is formed on thetarget blood vessel segment or grafted lumen thereby inhibitingneo-intimal hyperplasia.

In regard to diseases such as cancer, most therapeutic agents used todate have generalized systemic effects on the patient, not onlyaffecting the cancer cells, but any dividing cell in the body due to theuse of drugs in conventional oral or intravenous formulations. Yet inmany cases, systemic administration is not effective due to the natureof the disease that is in need of treatment and the properties of thedrug such as solubility, in vivo stability, bioavailability, etc. Uponsystemic administration, the drug is conveyed by blood circulation anddistributed into body areas including normal tissues. At diseased sites,the drug concentration is first low and ineffective which frequentlyincreases to toxic levels, while in non-diseased areas, the presence ofthe drug causes undesired side effect. In certain instances, drugs arereadily susceptible to metabolic degradation after being administered.Therefore, drug dose is often increased to achieve pharmacologicalefficacy and prolong duration, which causes increased systemic burden tonormal tissues as well as cost concern for the patient. In otherinstances, the therapeutic potential of some potent drugs cannot befulfilled due to their toxic side effects.

Therefore, much effort has been made to improve efficacy and targetingof drug delivery systems. For example, the use of liposomes to deliverdrugs has been advantageous in that, in general, they increase the drugcirculation time in blood, reduce side effects by limiting theconcentration of free drug in the bloodstream, decrease drugdegradation, prolong the therapeutic effect after each administration,reduce the need for frequent administration, and reduce the amount ofdrug needed. However, liposome systems that are currently available showlimited efficiency of delivering drugs to target sites in vivo. See Kayeet al., 1979, Poznansky et al. 1984, U.S. Pat. Nos. 5,043,165, and4,920,016.

To yield highly efficient delivery of therapeutic compounds, viralvectors able to incorporate transgenic DNA have been developed, yet thenumber of successful clinical applications has been limited. Despite thenumber of successes in vitro and in animal models, gene transfertechnology is therefore proposed to marry with cell therapy. The ex vivotransfer of gene combinations into a variety of cell types will likelyprove more therapeutically feasible than direct in vivo vector transfer.See Kohn et al., 1987, Bilbao et al., 1997, and Giannoukakis et al.2003.

More recently local drug delivery vehicles such as drug eluting stents(DES) have been developed. See U.S. Pat. Nos. 6,273,913, 6,258,121, and6,231,600. However, drug eluting stents of the prior art are limited bymany factors such as, the type of drug, the amount of drug to bereleased and the amount of time it takes to release the drug. Otherfactors which need to be considered in regards to drug eluting stentsare the drug interactions with other stent coating components, such aspolymer matrices, and individual drug properties includinghydrophobicity, molecular weight, intactness and activity aftersterilization, as well as efficacy and toxicity. With respect to polymermatrices of drug eluting stents, one must consider the polymer type,polymer ratio, drug loading capability, and biocompatibility of thepolymer and the drug-polymer compatibility such as drugpharmacokinetics.

Additionally, the drug dose in a drug eluting stent is pre-loaded and anadjustment of drug dose upon individual conditions and need cannot beachieved. In regard to drug release time, drug eluting stents instantlystart to release the drug upon implantation and an ideal real-timerelease cannot be achieved.

It is therefore a long-felt need to develop an efficient systemic andlocal drug delivery system to overcome limitations of current availabletechniques. The present invention provides a system for the delivery oftherapeutic agents locally or systemically in a safe and controlledmanner.

SUMMARY OF INVENTION

It is an object of the invention to provide a therapeutic, drug deliverysystem and method for treating diseases in a patient. The therapeutic ordrug delivery system comprises a medical device with a coating composedof a matrix comprising at least one type of ligand for recognizing andbinding target cells such as progenitor endothelial cells orgenetically-altered mammalian cells and genetically-altered mammaliancells which have been at least singly or dually-transfected.

The medical device of the invention can be any device that isimplantable into a patient. For example, in one embodiment the device isfor insertion into the lumen of a blood vessels or a hollowed organ,such as stents, stent grafts, heart valves, catheters, vascularprosthetic filters, artificial heart, external and internal leftventricular assist devices (LVADs), and synthetic vascular grafts, forthe treatment of diseases such as cancer, vascular diseases, including,restenosis, artherosclerosis, thrombosis, blood vessel obstruction, orany other applications additionally covered by these devices.

In one embodiment, the coating on the present medical device comprises abiocompatible matrix and at least one type of substance or ligand, whichspecifically recognize and bind target cells such as progenitorendothelial cells such as in the prevention or treatment of restenosis,or genetically-altered mammalian cells, onto the surface of the device,such as in the treatment of blood vessel remodeling and cancer.

Additionally, the coating of the medical device may optionally compriseat least an activating compound for regulating the expression andsecretion of the engineered genes of the genetically-altered cells.Examples of activator stimulatory compounds, include but is not limitedto chemical moieties, and peptides, such as growth factors. Inembodiments of the invention when the coating comprises at least onecompound, the activator molecule or compound may function to stimulatethe cells to express and/or secrete at least one therapeutic substancefor the treatment of disease.

In one embodiment, the coating on the medical device comprises abiocompatible matrix which comprises an outer surface for attaching atherapeutically effective amount of at least one type of ligand such asan antibody, antibody fragment, or a combination of the antibody and theantibody fragment, or at least one type of molecule for binding theengineered marker on the surface of the genetically-modified cell. Thepresent antibody or antibody fragment recognizes and binds an antigen orthe specific genetically-engineered cell surface marker on the cellmembrane or surface of target cells so that the cells are immobilized onthe surface of the device. In one embodiment, the coating may optionallycomprise an effective amount of at least one compound for stimulatingthe immobilized progenitor endothelial cells to either accelerate theformation of a mature, functional endothelium if the target cells arecirculating progenitor cells, or to stimulate the bound cells to expressand secrete the desired gene products if the target aregenetically-altered cells on the surface of the medical device.

The medical device of the invention can be any device used forimplanting into an organ or body part comprising a lumen, and can be,but is not limited to, a stent, a stent graft, a synthetic vasculargraft, a heart valve, a catheter, a vascular prosthetic filter, apacemaker, a pacemaker lead, a defibrillator, a patent foramen ovale(PFO) septal closure device, a vascular clip, a vascular aneurysmoccluder, a hemodialysis graft, a hemodialysis catheter, anatrioventricular shunt, an aortic aneurysm graft device or components, avenous valve, a suture, a vascular anastomosis clip, an indwellingvenous or arterial catheter, a vascular sheath and a drug delivery port.The medical device can be made of numerous materials depending on thedevice. For example, a stent of the invention can be made of stainlesssteel, Nitinol (NiTi), or chromium alloy and biodegradable materials.Synthetic vascular grafts can be made of a cross-linked PVA hydrogel,polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene(ePTFE), porous high density polyethylene (HDPE), polyurethane, andpolyethylene terephthalate, or biodegradable materials.

The biocompatible matrix forming the coating of the present medicaldevice comprises without limitation a synthetic material such aspolyurethanes, segmented polyurethane-urea/heparin, poly-L-lactic acid,cellulose ester, polyethylene glycol, polyvinyl acetate, dextran andgelatin, and/or naturally-occurring material such as basement membranecomponents such as collagen, elastin, laminin, fibronectin, vitronectin,heparin, fibrin, cellulose, and amorphous carbon, or fullerenes.

In an embodiment of the invention, the medical device comprises abiocompatible matrix comprising fullerenes. In this embodiment, thefullerene can range from about C₂₀ to about C₁₅₀ in the number of carbonatoms, and more particularly, the fullerene is C₆₀ or C₇₀. The fullereneof the invention can also be arranged as nanotubes on the surface of themedical device.

In one embodiment of the invention, the ligand is applied to the bloodcontacting surface of the medical device and the ligand specificallyrecognizes and binds a desired component or epitope on the surface oftarget cells in the circulating blood. In one embodiment, the ligand isspecifically designed to recognize and bind only the genetically-alteredmammalian cell by recognizing only the genetically-engineered markermolecule on the cell membrane of the genetically-altered cells. Thebinding of the target cells immobilizes the cells on the surface of thedevice.

In one embodiment, the ligand on the surface of the medical device forbinding the genetically-altered cell is selected depending on thegenetically engineered cell membrane marker molecule. That is, theligand binds only to the cell membrane marker which is expressed by thecell from the extrachromosomal genetic material so that only thegenetically-modified cells bind to the surface of the medical device.For example, if the mammalian cell is an endothelial cell, the ligandcan be at least one type of antibody specifically raised against aspecific target epitope or marker molecule on the surface of the targetcell. In this aspect of the invention, the antibody can be a monoclonalantibody, a polyclonal antibody, a chimeric antibody, or a humanizedantibody which recognizes and binds only to the genetically-alteredendothelial cell by interacting with the surface marker molecule and,thereby modulating the adherence of the cells onto the surface of themedical device. The antibody or antibody fragment of the invention canbe covalently or noncovalently attached to the surface of the matrix, ortethered covalently by a linker molecule to the outermost layer of thematrix coating the medical device. In this embodiment of the invention,for example, the monoclonal antibodies can further comprises Fab orF(ab′)₂ fragments. The antibody fragment of the invention comprises anyfragment size, such as large and small molecules which retain thecharacteristic to recognize and bind the target antigen as the antibody.

In another embodiment, the antibody or antibody fragment of theinvention recognize and bind antigens with specificity for the mammalbeing treated and their specificity is not dependent on cell lineage. Inone embodiment, for example, in treating restenosis wherein the cellsmay not be genetically modified to contain specific cell membrane markermolecules, the antibody or fragment is specific for selecting andbinding circulating progenitor endothelial cell surface antigen such asCD133, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146),CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit ligand),Tie-2 and HAD-DR.

In another embodiment, the coating of the medical device comprises atleast one layer of a biocompatible matrix as described above, the matrixcomprising an outer surface for attaching a therapeutically effectiveamount of at least one type of small molecule of natural or syntheticorigin. The small molecule recognizes and interacts with, for example,progenitor endothelial cells in the treatment of restenosis, toimmobilize the cells on the surface of the device to form an endotheliallayer. The small molecules can be used in conjunction with the medicaldevice for the treatment of various diseases, and can be derived from avariety of sources such as cellular components such as fatty acids,proteins, nucleic acids, saccharides and the like and can interact withan antigen on the surface of a progenitor endothelial cell with the sameresults or effects as an antibody. In this aspect of the invention, thecoating on the medical device can further comprise a compound such as agrowth factor as described herewith in conjunction with the coatingcomprising an antibody or antibody fragment.

In one embodiment, the compound of the coating of the invention, forexample in treating restenosis, comprises any compound which stimulatesor accelerates the growth and differentiation of the progenitor cellinto mature, functional endothelial cells. In another embodiment, thecompound is for stimulating the genetically modified cells to expressand secrete the desired gene product. For example, a compound for use inthe invention may be a growth factor such as vascular endothelial growthfactor (VEGF), basic fibroblast growth factor, platelet-induced growthfactor, transforming growth factor beta 1, acidic fibroblast growthfactor, osteonectin, angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2),insulin-like growth factor, granulocyte-macrophage colony-stimulatingfactor, platelet-derived growth factor AA, platelet-derived growthfactor BB, platelet-derived growth factor AB and endothelial PAS protein1.

In another embodiment, for example when using genetically-alteredmammalian cells, the activating agents or compounds useful forstimulating the cells to express and secrete the genetically-engineeredgene products include, but are not limited to estrogen, tetracycline andother antibiotics, tamoxiphen, etc., and can be provided to the patientvia various routes of administration, such as through the skin via apatch and subcutaneously.

The invention also provides methods for treating a variety of diseases,such as vascular disease, cancer, blood vessel remodeling, severecoronary artery disease. artherosclerosis, restenosis, thrombosis,aneurysm and blood vessel obstruction. In one embodiment, the methodprovides an improvement over prior art methods as far as retaining orsealing the medical device insert to the vessel wall, such as a stent orsynthetic vascular graft, heart valve, abdominal aortic aneurysm devicesand components thereof, for establishing vascular homeostasis, andthereby preventing excessive intimal hyperplasia as in restenosis. Inthe present method of treating atherosclerosis, the artery may be eithera coronary artery or a peripheral artery such as the femoral artery.Veins can also be treated using the techniques and medical device of theinvention.

With respect to the treatment of restenosis, the invention also providesan engineered method for inducing a healing response. In one embodiment,a method is provided for rapidly inducing the formation of a confluentlayer of endothelium in the luminal surface of an implanted device in atarget lesion of an implanted vessel, in which the endothelial cellsexpress nitric oxide synthase and other anti-inflammatory andinflammation-modulating factors. The invention also provides a medicaldevice which has increased biocompatibility over prior art devices, anddecreases or inhibits tissue-based excessive intimal hyperplasia andrestenosis by decreasing or inhibiting smooth muscle cell migration,smooth muscle cell differentiation, and collagen deposition along theinner luminal surface at the site of implantation of the medical device.

In an embodiment of the invention, a method for coating a medical devicecomprises the steps of: applying at least one layer of a biocompatiblematrix to the surface of the medical device, wherein the biocompatiblematrix comprises at least one component selected from the groupconsisting of a polyurethane, a segmented polyurethane-urea/heparin, apoly-L-lactic acid, a cellulose ester, a polyethylene glycol, apolyvinyl acetate, a dextran, gelatin, collagen, elastin, laminin,fibronectin, vitronectin, heparin, fibrin, cellulose and carbon andfullerene, and applying to the biocompatible matrix, simultaneously orsequentially, a therapeutically effective amounts of at least one typeof antibody, antibody fragment or a combination thereof, and at leastone compound which stimulates endothelial cell growth anddifferentiation.

The invention further provides a method for treating vascular disease ina mammal comprises implanting a medical device into a vessel or tubularorgan of the mammal, wherein the medical device is coated with (a) abiocompatible matrix, (b) therapeutically effective amounts of at leastone type of antibody, antibody fragment or a combination thereof, and(c) at least one compound; wherein the antibody or antibody fragmentrecognizes and binds an antigen on a progenitor endothelial cell surfaceso that the progenitor endothelial cell is immobilized on the surface ofthe matrix, and the compound is for stimulating the immobilizedprogenitor endothelial cells to form an endothelium on the surface ofthe medical device.

In one embodiment of the invention, a therapeutic/drug delivery systemfor treating a disease in a patient is also provided. The therapeutic ordrug delivery system comprises genetically-altered mammalian cells,comprising exogenous nucleic acid encoding a genetically-engineered cellmembrane marker and at least one therapeutic gene product, and a medicaldevice for implantation into a patient. In one embodiment, the geneticengineered cells are transfected in vitro with an appropriatetransfection vector comprising the exogenous genetic material forproviding the desired genes to the cells. In this embodiment of theinvention the cells can be any mammalian cell, either autologous,allogenic or xenogenic, such as endothelial cells, fibroblasts,myoblasts and the like. In this embodiment of the invention, the medicaldevice is coated with a biocompatible matrix comprising a ligand whichbinds only to the genetically-altered mammalian cells by way binding thegenetically-engineeered cell membrane marker on the surface of thecells.

In the therapeutic and/or drug delivery system of this embodiment, thegenetically-altered cells are provided with exogenous genetic materialto introduce at least one desired gene which encodes a cell surfacemarker and at least one gene which encodes a therapeutic gene product.The system optionally comprises a signal system, such as an activatingcompound or molecule for stimulating the genetically-altered mammaliancells to express and/or secrete the desired gene product and/or themarker gene.

Thus, in one embodiment of the invention, the exogenous genetic materialfor introducing into mammalian cells is engineered to encode a cellmembrane marker which specifically binds to the ligand on the device.For example, if the device is for implantation in a blood vessel lumen,the exogenous genetic material encodes a cell membrane marker not foundin any cell circulating in the blood stream, other than thegenetically-engineered cells provided to the patient.

It is an object of the invention to provide a coated medical devices andmethods for the treatment of a variety of diseases such as vasculardisease including but not limited to atherosclerosis, cancer, andrheumatoid arthritis. The medical device of the invention comprises acoating for the specific in vivo capturing and immobilization ofgenetically-altered mammalian cells which are introduced, simultaneouslyor sequentially, into the patient upon implantation of the coatedmedical device.

It is an object of the invention to provide the immobilizedgenetically-altered cells which express and/or secrete at least one typeof substance or therapeutic agent for the treatment of a specificdisease. In this aspect of the invention, for example in the treatmentof cancer, the cells, e.g., endothelial cells are genetically-altered byintroducing exogenous genetic material into the cells. In oneembodiment, the genetic material is introduced into the nucleus of thecells and is DNA, such as extrachromosomal DNA. The extrachromosomal DNAmay be a vector such an adenoviral vector, a plasmid such as a nakedplasmid, linear or short DNA, and the like. In one embodiment, the DNAcomprises regulatory/expression cassette for controlling the expressionof the desired marker and/or therapeutic genes. In one embodiment, theregulatory cassette may comprise regulatory elements for constitutiveexpression of the therapeutic genes or may comprise elements that can becontrolled or expressed as needed by the patient.

In one embodiment, the medical device for implantation into the patientcomprises a coating; the coating comprises a matrix bearing at least onetype of ligand, which recognizes and binds target cells. In theembodiment where the cells are genetically-altered, the ligand onlyrecognizes a specific cell membrane marker which is engineered into thecells. Thus in this embodiment of the invention, such ligand onlyrecognizes the genetically-altered mammalian cells introduced into thepatient, and the genetically-altered mammalian cells bind to saidmedical device and express and secrete said at least one therapeuticgene product.

In another embodiment, the therapeutic or drug delivery system mayfurther comprise an activating molecule for stimulating saidgenetically-altered mammalian cells to express and/or secrete thedesired therapeutic gene products. In this aspect of the invention, acompound such as a chemical stimulus or a peptide can be provided to thepatient via an oral route, a thermal patch, intravenously, intradermallyand the like. In this embodiment, the genetically-altered mammaliancells may be autogenic or xenogenic, such as mature endothelial cells,fibroblasts, muscle cells, epithelial cells, etc. exogenous nucleic acidis extrachromosomal DNA. In one embodiment, the DNA is provided in theform of a vector, such as an adenovirus vector, naked plasmid DNA,linear DNA and the like. In one embodiment, the extrachromosomal DNAcomprises a regulatory cassette, a gene which encodes a cell membraneantigen and at least one gene which encodes a peptide for treating adisease. In one aspect of the invention, the cell membrane specific geneencodes, for example, an osteogenic or a prostatic cell membraneprotein.

In one embodiment of the invention, the extrachromosomal geneticmaterial comprises a gene which encodes the therapeutic/drug product,such as vascular endothelial growth factor and angiogenin for use inblood vessel remodeling, anti-angiogenic factor in the treatment ofcancer.

In another embodiment of the invention, a method for treating disease ina patient is provided. The method comprises:

-   -   providing genetically-altered mammalian cells to the patient;        comprising exogenous nucleic acid encoding a        genetically-engineered cell membrane marker and at least one        therapeutic gene product;    -   implanting a medical device comprising a coating into the        patient; the coating comprising a matrix bearing at least one        ligand, wherein the ligand recognizes and binds the        genetically-engineered cell membrane marker on the        genetically-altered mammalian cells, and wherein the        genetically-altered mammalian cells bind to the medical device        and express and secrete the therapeutic gene product. In an        embodiment of the invention, the therapeutic gene and gene        product comprises, for example, vascular endothelial growth        factor, angiogenin, anti-angiogenic factor, fibroblast growth        factor.

The invention also provides a method for treating disease in a patient,the method comprises: providing genetically-altered mammalian cells tothe patient; implanting a medical device into the patient; wherein themedical device comprises a coating which comprises a matrix bearing atleast one ligand, wherein the ligand specifically recognizes and bindsat least one receptor on the genetically-altered mammalian cells, andwherein the genetically-altered mammalian cells bind to the medicaldevice and comprise exogenous nucleic acid for expressing and secretinga therapeutic gene product.

In another embodiment, a method for recruiting cells to a bloodcontacting surface in vivo is provided. The method comprises providing ablood contacting surface positioned in the blood stream of a subject,said blood contacting surface configured to recruit target cellscirculating in the blood stream of the subject to the blood contactingsurface; and recruiting the target cells to the blood contactingsurface. In this embodiment, the blood contacting surface comprises theluminal surface of a medical device implanted into the subject. In thisembodiment of the invention, The recruited target cells on the bloodcontacting surface, for example, a stent or graft, canself-endothelialize the surface of the device in restoring normalendothelium at a site of blood vessel injury. The blood contactingsurface can be a biodegradable scaffolding or can be coated with abiodegradable, biocompatible material. In this aspect of the invention,the biodegradable scaffolding when implanted into a blood vesselundergoes in situ degradation and the neo-endothelium formed on theluminal surface of the device restores the blood vessel continuitythrough the injured site so as to form a functional neo-vessel.

In another embodiment, the invention comprises prosthesis, comprising:(a) a support member having an exterior surface and a blood contactingsurface; (b) a first layer of a cross-linked polymeric compound coatedonto said blood contacting surface of said support member; and, (c) asecond layer coated on said first layer, said second layer comprising atleast one ligand having an affinity for a targeted cell in vivo.

In another embodiment, a method for generating a self-endothelializinggraft in vivo, the method comprising: (a) providing a scaffoldingconfigured to function as a vascular graft, said scaffolding having alumen surface and exterior surface, said lumen surface comprisingligands specific for binding to endothelial progenitor cells; (b)implanting said scaffolding into a blood vessel of a subject; and (c)recruiting circulating endothelial progenitor cells to said lumensurface of said scaffolding to form a neo-endothelium.

In yet another embodiment, a method for generating aself-endothelializing graft in situ, the method comprising: (a)providing a prosthetic structure having a surface exposed to circulatingblood; (b) implanting the prosthetic structure into a subject; and (c)recruiting circulating endothelial progenitor cells from the blood tothe surface of the prosthetic structure to form a neo-endotheliumthereon.

In another embodiment, a method for generating a self-endothelializinggraft in situ, the method comprising: (a) providing a biodegradablescaffolding configured to function as a temporary vascular graft, thescaffolding having a lumen surface and exterior surface; (b) implantingthe biodegradable scaffolding into a blood vessel; (c) recruitingcirculating progenitor endothelial cells to the lumen surface of thebiodegradable scaffolding to form a neo-endothelium; (d) encapsulatingthe exterior surface of the scaffolding by vascular tissue to form anexterior hemostatic vascular structure; and (e) degrading thebiodegradable scaffolding under in vivo conditions within a time framewhich allows the neo-endothelium and the exterior vascular structure toform a functional neo-vessel.

In an embodiment, a biodegradable scaffolding for forming anendothelialized vascular graft in situ, the scaffolding comprising: (a)a porous biodegradable support member having a lumen and an exteriorsurface; and (b) the lumen surface comprising a first layer of at leastone species of a polymeric compound coated to the support member, andwherein the compound is cross-linked to itself with a cross-linkingagent that forms covalent bonds that are subject to enzymatic cleavageor non-enzymatic hydrolysis under in vivo conditions.

In another embodiment, a method for generating a self-endothelializinggraft in situ, the method comprising: (a) providing a prostheticstructure, having a surface exposed to circulating blood; (b) implantingthe prosthetic structure into a subject; and (c) recruiting circulatingprogenitor endothelial cells from the blood to the surface of theprosthetic structure to form a neo-endothelium.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic representation of an antibody tethered covalentlyto the matrix by a cross-linking molecule. FIG. 1B shows a diagram ofthe C₆₀O molecule anchoring the matrix. FIG. 1C depicts a schematicrepresentation of a stent coated with the matrix of the inventionshowing how cells interact with ligands on the surface of a device.

FIG. 2A is a phase contrast micrograph of progenitor endothelial cellsadhered to a fibronectin-coated slide containing cells isolated byenriched medium. FIG. 2B is a phase contrast micrograph of progenitorendothelial cells adhered to a fibronectin-coated slide containing cellsisolated by anti-CD34 antibody coated magnetic beads. FIGS. 2D and 2Fare micrographs of the progenitor endothelial cells which had beenincubated for 7 days and stained with PI nuclear stain. As seen in thesefigures, the cells express mature endothelial cell markers as shown bythe antibody fluorescence for Tie-2 (FIGS. 2E and 2G) and VEGFR-2 (FIG.2C) antibody reactivity.

FIGS. 3A and 3B are photographs of a 2% agarose gel stained withethidium bromide of a semiquantitative RT-PCR for endothelial nitricoxide synthatase, eNOS and glyceraldehyde phosphate dehydrogenase,GAPDH. After 3 days (FIG. 3B) and 7 days (FIG. 3A) in culture onfibronectin-coated slides, the progenitor endothelial cells begin toexpress eNOS mRNA.

FIGS. 4A-4E are photomicrographs of HUVECs attached to the CMDx andanti-CD34 antibody (4A); gelatin and anti-CD34 antibody (4B); barestainless steel disc (4C); CMDx coated (4D) and gelatin coated (4E)stainless steel disc which were incubated with HUVEC cell and stainedwith propidium iodide.

FIGS. 5A-5C are photomicrographs of a control, coated with CMDx withoutantibody which were incubated with the white cell fraction of humanblood. The cells were stained with propidium iodide and FITC labeledanti-KDR antibody. FIGS. 5D-5F are photomicrographs of control stainlesssteel discs coated with gelatin without antibody bound to its surfacewhich were incubated with the white cell fraction of human blood. Thecells were stained with propidium iodide and FITC labeled anti-KDRantibody.

FIGS. 6A-6C are photomicrographs of stainless steel discs coated withCMDx matrix with anti-CD34 antibody bound to its surface which wereincubated with the HUVECs. The cells were stained with propidium iodideand FITC labeled anti-KDR antibody. FIGS. 6D-6F are photomicrographs ofstainless steel discs coated with gelatin matrix with antibody bound toits surface, which were incubated with HUVECS. The cells were stainedwith propidium iodide and FITC labeled anti-KDR antibody.

FIG. 7 is a photomicrograph of stainless steel discs coated with CMDxmatrix with antibody bound to its surface, which was incubated withprogenitor cells for 24 hours. The cells were stained with propidiumiodide and FITC labeled anti-KDR antibody.

FIGS. 8A and 8B are photomicrographs of a stainless steel disc coatedwith CMDx matrix containing anti-CD34 antibody bound to its surfaceincubated with progenitor cells for 7 days. The cells were stained withpropidium iodide and FITC labeled anti-KDR antibody.

FIGS. 9A and 9B photomicrograph of a stainless steel disc coated withCMDx matrix containing anti-CD34 antibody bound to its surface incubatedwith progenitor cells for 7 days. The cells were stained with propidiumiodide and FITC labeled anti-Tie-2 antibody.

FIGS. 10A-10C are phase contrast photomicrographs of stainless steelCMDx coated discs incubated with progenitor cells for 3 weeks inendothelial growth medium which show mature endothelial cells.

FIG. 11 is schematic diagram of a functional fullerene coated stentsurface of the invention binding a progenitor cell.

FIGS. 12A-12D are photomicrographs of fullerene-coated samples withoutor with anti-CD34 antibody. The samples were incubated with a humanwhite blood cell fraction and stained with Propidium iodide and FITClabeled anti-VEGFR-2 antibody.

FIGS. 13A-13D are photomicrographs of histological cross-sections ofcoronary artery explants which had been implanted for 4 weeks with abare stainless steel stent (FIGS. 13A and 13C) and a fullerene-coatedsample (FIGS. 13B and 13D) taken at low and high magnification. Thesections were stained with hematoxylin-eosin stain.

FIGS. 14A-14G are scanning electron micrographs of stent explants 1 and48 hours after implantation in male Yorkshire swine. Explants ofdextran-coated (FIG. 14A) and dextran/anti-CD34 antibody-coated (14B)stents at 1 hour after implantation. FIGS. 14C and 14D show explants ofcontrol samples and FIGS. 14E-G are dextran/anti-CD34 antibody-coatedstents at 48 hours after implantation. FIGS. 14H-14M are histologicalphotomicrographs of cross-sections through coronary arteries of explantsfrom male Yorkshire swine which were implanted for 4 weeks: uncoated(Bare stainless steel) (14H and 14I), dextran-coated control (14J and14K), and dextran/anti-CD34 antibody-coated (14L and 14M).

FIGS. 15A, 15B and 15C are, respectively, fluorescent photomicrographsof 48 hours explants of a dextran-plasma-coated stent without antibodyon its surface, and a dextran-plasma-coated/anti-CD34 antibody-coatedstent of 18 mm in length.

FIGS. 16A and 16B are photomicrographs of a Propidium iodide andanti-lectin/FITC-conjugated sample.

DETAILED DESCRIPTION

The present invention provides a coated, implantable medical device suchas a stent, methods and compositions for coating the medical device, andmethods of treating vascular disease with the coated medical device.FIGS. 1A-1C show a schematic representation of the surface coat of amedical device of the invention. The coat on the medical devicecomprises a biocompatible matrix for promoting the formation of aconfluent layer of endothelial cells on the surface of the device toinhibit excessive intimal hyperplasia, and thereby preventing restenosisand thrombosis. In one embodiment, the matrix comprises a synthetic ornaturally-occurring material in which a therapeutically effective amountof at least one type of antibody that promotes adherence of endothelial,progenitor or stem cells to the medical device, and at least onecompound such as a growth factor, which stimulates endothelial cellgrowth and differentiation. Upon implantation of the device, the cellsthat adhere to the surface of the device transform into a mature,confluent, functional layer of endothelium on the luminal surface of themedical device. The presence of a confluent layer of endothelial cellson the medical device reduces the occurrence of restenosis andthrombosis at the site of implantation.

As used herein, “medical device” refers to a device that is introducedtemporarily or permanently into a mammal for the prophylaxis or therapyof a medical condition. These devices include any that are introducedsubcutaneously, percutaneously or surgically to rest within an organ,tissue or lumen of an organ, such as an artery, vein, ventricle, oratrium of the heart. Medical devices may include stents, stent grafts,covered stents such as those covered with polytetrafluoroethylene(PTFE), expanded polytetrafluoroethylene (ePTFE), or other natural orsynthetic coverings, or synthetic vascular grafts, artificial heartvalves, artificial hearts and fixtures to connect the prosthetic organto the vascular circulation, venous valves, abdominal aortic aneurysm(AAA) grafts, inferior venal caval filters, permanent drug infusioncatheters, embolic coils, embolic materials used in vascularembolization (e.g., cross-linked PVA hydrogel), vascular sutures,vascular anastomosis fixtures, transmyocardial revascularization stentsand/or other conduits.

Coating of the medical device with the compositions and methods of thisinvention stimulates the development of a confluent mammalian cell layersuch as an endothelial cell layer on the surface of the medical device,thereby preventing restenosis as well as modulating the local chronicinflammatory response and thromboembolic complications that result fromimplantation of the medical device.

The matrix coating the medical device can be composed of syntheticmaterial, such as polymeric gel foams, such as hydrogels made frompolyvinyl alcohol (PVA), polyurethane, poly-L-lactic acid, celluloseester or polyethylene glycol. In one embodiment, very hydrophiliccompounds such as dextran compounds can comprise the synthetic materialfor making the matrix. In another embodiment, the matrix is composed ofnaturally occurring materials, such as collagen, fibrin, elastin, oramorphous carbon. The matrix may comprise several layers with a firstlayer being composed of synthetic or naturally occurring materials and asecond layer composed of antibodies. The layers may be orderedsequentially, with the first layer directly in contact with the stent orsynthetic graft surface and the second layer having one surface incontact with the first layer and the opposite surface in contact withthe vessel lumen.

The matrix further comprises at least a growth factor, cytokine or thelike, which stimulates endothelial cell proliferation anddifferentiation. For example, vascular endothelial cell growth factor(VEGF) and isoforms, basic fibroblast growth factor (bFGF),platelet-induced growth factor (PIGF), transforming growth factor beta 1(TGF. b1), acidic fibroblast growth factor (aFGF), osteonectin,angiopoietin 1, angiopoietin 2, insulin-like growth factor (ILGF),platelet-derived growth factor AA (PDGF-AA), platelet-derived growthfactor BB (PDGF-BB), platelet-derived growth factor AB (PDGF-AB),granulocyte-macrophage colony-stimulating factor (GM-CSF), and the like,or functional fragments thereof can be used in the invention.

In another embodiment, the matrix may comprise fullerenes, where thefullerenes range from about C₂₀ to about C₁₅₀ in carbon number. Thefullerenes can also be arranged as nanotubes, that incorporate moleculesor proteins. The fullerene matrix can also be applied to the surface ofstainless steel, PTFE, or ePTFE medical devices, which layer is thenfunctionalized and coated with antibodies and growth factor on itssurface. Alternatively, the PTFE or ePTFE can be layered first on, forexample, a stainless steel medical device followed by a second layer offullerenes and then the antibodies and the growth factor are added.

The matrix may be noncovalently or covalently attached to the medicaldevice. Antibodies and growth factors can be covalently attached to thematrix using hetero- or homobifunctional cross-linking reagents. Thegrowth factor can be added to the matrix using standard techniques withthe antibodies or after antibody binding.

As used herein, the term “antibody” refers to one type of monoclonal,polyclonal, humanized, or chimeric antibody or a combination thereof,wherein the monoclonal, polyclonal, humanized or chimeric antibody bindsto one antigen or a functional equivalent of that antigen. The termantibody fragment encompasses any fragment of an antibody such as Fab,F(ab′)₂, and can be of any size, i.e., large or small molecules, whichhave the same results or effects as the antibody. (An antibodyencompasses a plurality of individual antibody molecules equal to6.022×10²³ molecules per mole of antibody).

In an aspect of the invention, a stent or synthetic graft of theinvention is coated with a biocompatible matrix comprising antibodiesthat modulate adherence of circulating progenitor endothelial cells tothe medical device. The antibodies of the invention recognize and bindthe target mammalian cells such as therapeutic cells or progenitorendothelial cells by their specific surface antigens which cells arecirculating in the blood stream so that the cells are immobilized on thesurface of the device. In one embodiment, for example, the antibodiescomprise monoclonal antibodies reactive (recognize and bind) withprogenitor endothelial cell surface antigens, or a progenitor or stemcell surface antigen, such as vascular endothelial growth factorreceptor-1, -2 and -3 (VEGFR-1, VEGFR-2 and VEGFR-3 and VEGFR receptorfamily isoforms), Tie-1, Tie2, CD34, Thy-1, Thy-2, Muc-18 (CD146), CD30,stem cell antigen-1 (Sca-1), stem cell factor (SCF or c-Kit ligand),CD133 antigen, VE-cadherin, P1H12, TEK, CD31, Ang-1, Ang-2, or anantigen expressed on the surface of progenitor endothelial cells.

In one embodiment, a single type of antibody that reacts with oneantigen can be used as the ligand for binding the target cells.Alternatively, a plurality of different antibodies directed againstvarious surface antigens can be mixed together and added to the matrix.In another embodiment, a cocktail of monoclonal antibodies is used toincrease the rate of a cell monolayer, such as endothelium formation, bytargeting specific cell surface antigens. In this aspect of theinvention, for example, anti-CD34 and anti-CD133 are used in combinationand attached to the surface of the matrix on a stent.

As used herein, a “therapeutically effective amount of the antibody”means the amount of an antibody that promotes adherence of endothelial,progenitor or stem cells to the medical device. The amount of anantibody needed to practice the invention varies with the nature of theantibody used. For example, the amount of an antibody used depends onthe binding constant between the antibody and the antigen against whichit reacts. It is well known to those of ordinary skill in the art how todetermine therapeutically effective amounts of an antibody to use with aparticular antigen.

As used herein, the term “compound” refers to any substance whichstimulates cells such as genetically-altered mammalian cells to expressand/or secrete the therapeutic gene product.

As used herein, the term “growth factor” refers to a peptide, protein,glycoprotein, lipoprotein, or a fragment or modification thereof, or asynthetic molecule, which stimulates endothelial, stem or progenitorcells to grow and differentiate into mature, functional endothelialcells. Mature endothelial cells express nitric oxide synthetase, therebyreleasing nitric oxide into the tissues. Table 1 below lists some of thegrowth factors that can be used for coating the medical device. TABLE 1Endothelial cell Growth Factor specific Acidic fibroblast growth factor(aFGF) No Basic fibroblast growth factor (bFGF) No Fibroblast growthfactor 3 (FGF-3) No Fibroblast growth factor 4 (FGF-4) No Fibroblastgrowth factor 5 (FGF-5) No Fibroblast growth factor 6 (FGF-6) NoFibroblast growth factor 7 (FGF-7) No Fibroblast growth factor 8 (FGF-8)No Fibroblast growth factor 9 (FGF-9) No Angiogenin 1 Yes Angiogenin 2Yes Hepatocyte growth factor/scatter factor (HGF/SF) No Platelet-derivedgrowth factor (PDE-CGF) Yes Transforming growth factor-α (TGF-α) NoTransforming growth factor-β (TGF-β) No Tumor necrosis factor-α (TNF-α)No Vascular endothelial growth factor 121 (VEGF 121) Yes Vascularendothelial growth factor 145 (VEGF 145) Yes Vascular endothelial growthfactor 165 (VEGF 165) Yes Vascular endothelial growth factor 189 (VEGF189) Yes Vascular endothelial growth factor 206 (VEGF 206) Yes Vascularendothelial growth factor B (VEGF-B) Yes Vascular endothelial growthfactor C (VEGF-C) Yes Vascular endothelial growth factor D (VEGF-D) YesVascular endothelial growth factor E (VEGF-E) Yes Vascular endothelialgrowth factor F (VEGF-F) Yes Placental growth factor Yes Angiopoietin-1No Angiopoietin-2 No Thrombospondin (TSP) No Proliferin Yes Ephrin-Al(B61) Yes E-selectin Yes Chicken chemotactic and angiogenic factor(cCAF) No Leptin Yes Heparin affinity regulatory peptide (HARP) NoHeparin No Granulocyte colony stimulating factor No Insulin-like growthfactor No Interleukin 8 No Thyroxine No Sphingosine 1-phosphate No

As used herein, the term “VEGF” means any of the isoforms of thevascular endothelium growth factor listed in Table 1 above unless theisoform is specifically identified with its numerical or alphabeticalabbreviation.

As used herein, the term “therapeutically effective amounts of growthfactor” means the amount of a growth factor that stimulates or inducesendothelial, progenitor or stem cells to grow and differentiate, therebyforming a confluent layer of mature and functional endothelial cells onthe luminal surface of the medical device. The amount of a growth factorneeded to practice the invention varies with the nature of the growthfactor used and binding kinetics between the growth factor and itsreceptor. For example, 100 μg of VEGF has been shown to stimulate theadherence of endothelial cells on a medical device and form a confluentlayer of epithelium. It is well known to those of ordinary skill in theart how to determine therapeutically effective amounts of a growthfactor to use to stimulate cell growth and differentiation ofendothelial cells.

As used herein, “intimal hyperplasia” is the undesirable increased insmooth muscle cell proliferation and/or matrix deposition in the vesselwall. As used herein “restenosis” refers to the recurrent narrowing ofthe blood vessel lumen. Vessels may become obstructed because ofrestenosis. After PTCA or PTA, smooth muscle cells from the media andadventitia, which are not normally present in the intima, proliferateand migrate to the intima and secrete proteins, forming an accumulationof smooth muscle cells and matrix protein within the intima. Thisaccumulation causes a narrowing of the lumen of the artery, reducingblood flow distal to the narrowing. As used herein, “inhibition ofrestenosis” refers to the inhibition of migration and proliferation ofsmooth muscle cells accompanied by prevention of protein secretion so asto prevent restenosis and the complications arising therefrom.

The subjects that can be treated using the medical device, methods andcompositions of this invention are mammals, or more specifically, ahuman, dog, cat, pig, rodent or monkey.

The methods of the present invention may be practiced in vivo or invitro.

The term “progenitor endothelial cell” refers to endothelial cells atany developmental stage, from progenitor or stem cells to mature,functional endothelial cells from bone marrow, blood or local tissueorigin and which are non-malignant.

For in vitro studies or use of the coated medical device, fullydifferentiated endothelial cells may be isolated from an artery or veinsuch as a human umbilical vein, while progenitor endothelial cells areisolated from peripheral blood or bone marrow. The endothelial cells arebound to the medical devices by incubation of the endothelial cells witha medical device coated with the matrix that incorporates an antibody, agrowth factor, or other agent that adheres to endothelial cells. Inanother embodiment, the endothelial cells can be transformed endothelialcells. The transfected endothelial cells contain vectors which expressgrowth factors or proteins which directly or indirectly inhibitthrombogenesis, restenosis, or any other therapeutic end.

In another embodiment, endothelial or any other type of stable mammaliancells are transfected with any mammalian expression vector that containsany cloned genes encoding proteins or peptides suitable for specificapplications. For example, the vector can be constructed consisting anexpression cassette comprising a gene encoding platelet derived growthfactor (PDGF), fibroblast growth factor (FGF), or nitric oxide synthase(NOS) and the expression cassette can be constructed using conventionalmethods, and supplies from commercially available sources. (See, forexample, mammalian expression vectors and transfection kits commerciallyavailable from Stratagene, San Diego, Calif.). For example, purifiedporcine progenitor endothelial cells are transfected with vascularendothelial growth factor (VEGF) using an adenoviral expression vectorexpressing the VEGF cDNA according to the methods of Rosengart et al.(Six-month assessment of a phase I trial of angiogenic gene therapy forthe treatment of coronary artery disease using direct intramyocardialadministration of an adenovirus vector expressing the VEGF121 cDNA. Ann.Surg. 230(4):466-470, 1999, incorporated herein by reference). In thisembodiment of the invention, the mammalian cells can be autologous,allogenic or xenogenic in origin. Once the cells are genetically-alteredby transfection of exogenous DNA or RNA expression cassettes comprisingthe desired genes, the cells can be grown using standard tissue culturetechniques. Samples of cells which express and secrete desired genes canbe stored frozen in liquid nitrogen using standard techniques. Frozenare regrown using standard tissue culture techniques prior to use. Thegenetically-altered cells are administered to the patient at the time ofimplantation of the device either locally at the implant site, orintravenously or intra-arterially into the patient, preferably after thecoated medical device is implanted. Transformed cells for use in theinvention can further comprise a marker or reporter gene for theaccurate detection and identification of the cells prior to celladministration to the patient.

The methods of treatment of vascular disease of the invention can bepracticed on any artery or vein. Included within the scope of thisinvention is atherosclerosis of any artery including coronary,infrainguinal, aortoiliac, subclavian, mesenteric and renal arteries.Other types of vessel obstructions, such as those resulting from adissecting aneurysm are also encompassed by the invention.

The method of treating a mammal with vascular disease comprisesimplanting a coated medical device into the patient's organ or vessel,for example, in the case of a coated stent during angioplasty. Once insitu, progenitor endothelial cells are captured on the surface of thecoated stent by the recognition and binding of antigens on theprogenitor cell surface by the antibody present on the coating. Once theprogenitor cell is adhered to the matrix, the growth factor on thecoating promotes the newly-bound progenitor endothelial cells to growand differentiate and form a confluent, mature and functionalendothelium on the luminal surface of the stent. Alternatively, themedical device is coated with the endothelial cells in vitro beforeimplantation of the medical device using progenitor, stem cells, ormature endothelial cells isolated from the patient's blood, bone marrow,or blood vessel. In either case, the presence of endothelial cells onthe luminal surface of the medical device inhibits or prevents excessiveintimal hyperplasia and thrombosis.

Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) are obtained fromumbilical cords according to the methods of Jaffe, et al., J. Clin.Invest., 52:2745-2757,1973, incorporated herein by reference and wereused in the experiments. Briefly, cells are stripped from the bloodvessel walls by treatment with collagenase and cultured ingelatin-coated tissue culture flasks in M199 medium containing 10% lowendotoxin fetal calf serum, 90 ug/ml preservative-free porcine heparin,20 ug/ml endothelial cell growth supplement (ECGS) and glutamine.

Progenitor endothelial cells (EPC) are isolated from human peripheralblood according to the methods of Asahara et al. (Isolation of putativeprogenitor endothelial cells for angiogenesis. Science 275:964-967,1997, incorporated herein by reference). Magnetic beads coated withantibody to CD34 are incubated with fractionated human peripheral blood.After incubation, bound cells are eluted and can be cultured in EBM-2culture medium. (Clonetics, San Diego, Calif.). Alternatively enrichedmedium isolation can be used to isolate these cells. Briefly, peripheralvenous blood is taken from volunteers and the mononuclear cell fractionis isolated by density gradient centrifugation, and the cells are platedon fibronectin coated culture slides (Becton Dickinson) in EC basalmedium-2 (EBM-2) (Clonetics) supplemented with 5% fetal bovine serum,human VEGF-A, human fibroblast growth factor-2, human epidermal growthfactor, insulin-like growth factor-1, and ascorbic acid. EPCs are grownfor 7-days, with culture media changes every 48 hours. Cells arecharacterized by fluorescent antibodies to CD45, CD34, CD31, VEGFR-2,Tie-2, and E-selectin.

In another embodiment, mammalian cells are transfected with anyexpression cassette that contains any cloned gene that encodes proteinssuch as platelet derived growth factor (PDGF), fibroblast growth factor(FGF), or nitric oxide synthase (NOS) using conventional methods. (See,for example, mammalian expression vectors and transfection kitscommercially available from Stratagene, San Diego, Calif.). For example,purified porcine progenitor endothelial cells are transfected withvascular endothelial growth factor (VEGF) using an mammalian expressioncassette expressing the VEGF cDNA according to the methods of Rosengartet al. (Six-month assessment of a phase I trial of angiogenic genetherapy for the treatment of coronary artery disease using directintramyocardial administration of an adenovirus vector expressing theVEGF121 cDNA. Ann. Surg. 230(4):466-470 (1999), incorporated herein byreference).

Antibodies

Monoclonal antibodies useful in the method of the invention may beproduced according to the standard techniques of Kohler and Milstein(Continuous cultures of fused cells secreting antibody of predefinedspecificity. Nature 265:495-497, 1975, incorporated herein byreference), or can be obtained from commercial sources. Endothelialcells can be used as the immunogen to produce monoclonal antibodiesdirected against endothelial cell surface antigens.

Monoclonal antibodies directed against endothelial cells are prepared byinjecting HUVEC or purified progenitor endothelial cells into a mouse orrat. After a sufficient time, the mouse is sacrificed and spleen cellsare obtained. The spleen cells are immortalized by fusing them withmyeloma cells or with lymphoma cells, generally in the presence of anon-ionic detergent, for example, polyethylene glycol. The resultingcells, which include the fused hybridomas, are allowed to grow in aselective medium, such as HAT-medium, and the surviving cells are grownin such medium using limiting dilution conditions. The cells are grownin a suitable container, e.g., microtiter wells, and the supernatant isscreened for monoclonal antibodies having the desired specificity, i.e.,reactivity with endothelial cell antigens.

Various techniques exist for enhancing yields of monoclonal antibodiessuch as injection of the hybridoma cells into the peritoneal cavity of amammalian host which accepts the cells and then harvesting the asciticfluid. Where an insufficient amount of monoclonal antibody collects inthe ascitic fluid, the antibody is harvested from the blood of the host.Various conventional ways exist for isolation and purification ofmonoclonal antibodies so as to free the monoclonal antibodies from otherproteins and other contaminants.

Also included within the scope of the invention are useful bindingfragments of anti-endothelial cell monoclonal antibodies such as theFab, F(ab′)₂ of these monoclonal antibodies. The antibody fragments areobtained by conventional techniques. For example, useful bindingfragments may be prepared by peptidase digestion of the antibody usingpapain or pepsin.

Antibodies of the invention are directed to an antibody of the IgG classfrom a murine source; however, this is not meant to be a limitation. Theabove antibody and those antibodies having functional equivalency withthe above antibody, whether from a murine source, mammalian sourceincluding human, or other sources, or combinations thereof are includedwithin the scope of this invention, as well as other classes such asIgM, IgA, IgE, and the like, including isotypes within such classes. Inthe case of antibodies, the term “functional equivalency” means that twodifferent antibodies each bind to the same antigenic site on an antigen,in other words, the antibodies compete for binding to the same antigen.The antigen may be on the same or different molecule.

In one embodiment, monoclonal antibodies reacting with the endothelialcell surface antigen CD34 are used. Anti-CD34 monoclonal antibodiesattached to a solid support have been shown to capture progenitorendothelial cells from human peripheral blood. After capture, theseprogenitor cells are capable of differentiating into endothelial cells.(Asahara et al. 1997. Isolation of putative progenitor endothelial cellsfor angiogenesis. Science 275:964-967.) Hybridomas producing monoclonalantibodies directed against CD34 can be obtained from the American TypeTissue Collection. (Rockville, Md.). In another embodiment, monoclonalantibodies reactive with endothelial cell surface antigens such asVEGFR-1 and VEGFR-2, CD133, or Tie-2 are used. In the embodiment usinggenetically-altered cell, antibodies are produced against thegenetically engineered gene product using standard techniques in thesame manner as described above, and then applied to the blood contactingsurface of the medical device following matrix application.

Polyclonal antibodies reactive against endothelial cells isolated fromthe same species as the one receiving the medical device implant mayalso be used.

Stent

The term “stent” herein means any medical device which when inserted orimplanted into the lumen of a vessel expands the cross-sectional lumenof a vessel. The term “stent” includes, stents commercially availablemanufactured from stainless steel or other alloys which have been coatedby the methods of the invention; covered stents such as those coveredwith PTFE or ePTFE. In one embodiment, this includes stents deliveredpercutaneously to treat coronary artery occlusions or to sealdissections or aneurysms of the splenic, carotid, iliac and poplitealvessels. In another embodiment, the stent is delivered into a venousvessel. The stent can be composed of polymeric or metallic structuralelements onto which the matrix comprising the antibodies and thecompound, such as growth factors, is applied or the stent can be acomposite of the matrix intermixed with a polymer. For example, adeformable metal wire stent can be used, such as that disclosed in U.S.Pat. No. 4,886,062 to Wiktor, incorporated herein by reference. Aself-expanding stent of resilient polymeric material such as thatdisclosed in published international patent application WO91/12779“Intraluminal Drug Eluting Prosthesis”, incorporated herein byreference, can also be used. Stents may also be manufactured usingstainless steel, polymers, nickel-titanium, tantalum, gold,platinum-iridium, cobalt-based alloys or Elgiloy and MP35N and otherferrous materials. Stents are delivered through the body lumen on acatheter to the treatment site where the stent is released from thecatheter, allowing the stent to expand into direct contact with theluminal wall of the vessel. In another embodiment, the stent comprises abiodegradable stent (H. Tamai, pp 297 in Handbook of Coronary Stents,3rd Edition, Eds. P W Serruys and M J B Kutryk, Martin Dunitz (2000). Itwill be apparent to those skilled in the art that other self-expandingstent designs (such as resilient metal stent designs) could be used withthe antibodies, growth factors and matrices of this invention.

Synthetic Graft

The term “synthetic graft” means any artificial prosthesis havingbiocompatible characteristics. In one embodiment, the synthetic graftscan be made of polyethylene terephthalate (Dacron®, PET) orpolytetrafluoroehtylene (Teflon®, ePTFE). In another embodiment,synthetic grafts are composed of polyurethane, cross-linked PVAhydrogel, and/or biocompatible foams of hydrogels. In yet a thirdembodiment, a synthetic graft is composed of an inner layer of meshedpolycarbonate urethane and an outer layer of meshed polyethyleneterephthalate. It will be apparent to those skilled in the art that anybiocompatible synthetic graft can be used with the antibodies, growthfactors, and matrices of this invention. (Bos et al. 1998.Small-Diameter Vascular Prostheses: Current Status. Archives PhysioBiochem. 106:100-115, incorporated herein by reference). Syntheticgrafts can be used for end-to-end, end to side, side to end, side toside or intraluminal and in anastomosis of vessels or for bypass of adiseased vessel segments, for example, as abdominal aortic aneurysmdevices.

Matrix

(A) Synthetic Materials—The matrix that is used to coat the stent orsynthetic graft may be selected from synthetic materials such aspolyurethane, segmented polyurethane-urea/heparin, poly-L-lactic acid,cellulose ester, polyethylene glycol, cross-linked PVA hydrogel,biocompatible foams of hydrogels, or hydrophilic dextrans, such ascarboxymethyl dextran.

(B) Naturally Occurring Material—The matrix may be selected fromnaturally occurring substances such as collagen, fibronectin,vitronectin, elastin, laminin, heparin, fibrin, cellulose or carbon. Aprimary requirement for the matrix is that it be sufficiently elasticand flexible to remain unruptured on the exposed surfaces of the stentor synthetic graft.

(C) Fullerenes—The matrix may also comprise a fullerene (the term“fullerene” encompasses a plurality of fullerene molecules). Fullerenesare carbon-cage molecules. The number of carbon (C) molecules in afullerene species varies from about C₂₀ to about C₁₅₀. Fullerenes areproduced by high temperature reactions of elemental carbon or ofcarbon-containing species by processes well known to those skilled inthe art; for example, by laser vaporization of carbon, heating carbon inan electric arc or burning of hydrocarbons in sooting flames. (U.S. Pat.No. 5,292,813, to Patel et al., incorporated herein by reference; U.S.Pat. No. 5,558,903 to Bhushan et al., incorporated herein by reference).In each case, a carbonaceous deposit or soot is produced. From thissoot, various fullerenes are obtained by extraction with appropriatesolvents, such as toluene. The fullerenes are separated by knownmethods, in particular by high performance liquid chromatography (HPLC).Fullerenes may be synthesized or obtained commercially from DynamicEnterprises, Ltd., Berkshire, England or Southern Chemical Group, LLC,Tucker, Ga., or Bucky USA, Houston Tex.

Fullerenes may be deposited on surfaces in a variety of different ways,including, sublimation, laser vaporization, sputtering, ion beam, spraycoating, dip coating, roll-on brush coating as disclosed in U.S. Pat.No. 5,558,903, or by derivatization of the surface of the stent.

An important feature of fullerenes is their ability to form “activatedcarbon.” The fullerene electronic structure is a system of overlappingpi-orbitals, such that a multitude of bonding electrons arecooperatively presented around the surface of the molecule. (Chemicaland Engineerinq News, Apr. 8, 1991, page 59, incorporated herein byreference). As forms of activated carbon, fullerenes exhibit substantialvan der Waals forces for weak interactions. The adsorptive nature of thefullerene surface may lend itself to additional modifications for thepurpose of directing specific cell membrane interactions. For example,specific molecules that possess chemical properties that selectivelybind to cell membranes of particular cell types or to particularcomponents of cell membranes, e.g., lectins or antibodies, can beadsorbed to the fullerene surface. Attachment of different molecules tothe fullerene surface may be manipulated to create surfaces thatselectively bind various cell types, e.g., progenitor endothelial cells,epithelial cells, fibroblasts, primary explants, or T-cellsubpopulations. U.S. Pat. No. 5,310,669 to Richmond et al., incorporatedherein by reference; Stephen R. Wilson, Biological Aspects ofFullerenes, Fullerenes:Chemistry, Physics and Technology, Kadish et al.eds., John Wiley & Sons, New York 2000, incorporated herein byreference.

Fullerenes may also form nanotubes that incorporate other atoms ormolecules. (Liu et al. Science 280:1253-1256 (1998), incorporated hereinby reference). The synthesis and preparation of carbon nanotubes is wellknown in the art. (U.S. Pat. No. 5,753,088 to Olk et al., and U.S. Pat.No. 5,641,466 to Ebbsen et al., both incorporated herein by reference).Molecules such as proteins can also be incorporated inside carbonnanotubes. For example, nanotubes may be filled with the enzymes, e.g.,Zn₂Cd₂-metallothionein, cytochromes C and C3, and beta-lactamase aftercutting the ends of the nanotube. (Davis et al. Inorganica Chim. Acta272:261 (1998); Cook et al. Full Sci. Tech. 5(4):695 (1997), bothincorporated herein by reference).

Three dimensional fullerene structures can also be used. U.S. Pat. No.5,338,571 to Mirkin et al., incorporated herein by reference, disclosesthree-dimensional, multilayer fullerene structures that are formed on asubstrate surface by (i) chemically modifying fullerenes to provide abond-forming species; (ii) chemically treating a surface of thesubstrate to provide a bond-forming species effective to covalently bondwith the bond-forming species of the fullerenes in solution; and, (iii)contacting a solution of modified fullerenes with the treated substratesurface to form a fullerene layer covalently bonded to the treatedsubstrate surface.

(D) Application of the Matrix to the Medical Device

The matrix should adhere tightly to the surface of the stent orsynthetic graft. Preferably, this is accomplished by applying the matrixin successive thin layers. Alternatively, antibodies and growth factorsare applied only to the surface of the outer layer in direct contactwith the vessel lumen. Different types of matrices may be appliedsuccessively in succeeding layers. The antibodies may be covalently ornoncovalently coated on the matrix after application of the matrix tothe stent.

In order to coat a medical device such as a stent, the stent is dippedor sprayed with a liquid solution of the matrix of moderate viscosity.After each layer is applied, the stent is dried before application ofthe next layer. In one embodiment, a thin, paint-like matrix coatingdoes not exceed an overall thickness of 100 microns.

In one embodiment, the stent surface is first functionalized, followedby the addition of a matrix layer. Thereafter, the antibodies and thegrowth factor are coupled to the surface of the matrix. In this aspectof the invention, the techniques of the stent surface creates chemicalgroups which are functional. The chemical groups such as amines, arethen used to immobilize an intermediate layer of matrix, which serves assupport for the antibodies and the growth factor.

In another embodiment, a suitable matrix coating solution is prepared bydissolving 480 milligrams (mg) of a drug carrier, such as poly-D,L-lactid (available as R203 of Boehringer Inc., Ingelheim, Germany) in 3milliliters (ml) of chloroform under aseptic conditions. In principle,however, any biodegradable (or non-biodegradable) matrix that isblood-and tissue-compatible (biocompatible) and can be dissolved,dispersed or emulsified may be used as the matrix if, after application,it undergoes relatively rapid drying to a self-adhesive lacquer- orpaint-like coating on the medical device.

For example, coating a stent with fibrin is well known to one ofordinary skill in the art. In U.S. Pat. No. 4,548,736 issued to Mulleret al., incorporated herein by reference, fibrin is clotted bycontacting fibrinogen with thrombin. Preferably, the fibrin in thefibrin-containing stent of the present invention has Factor XIII andcalcium present during clotting, as described in U.S. Pat. No. 3,523,807issued to Gerendas, incorporated herein by reference, or as described inpublished European Pat. Application 0366564, incorporated herein byreference, in order to improve the mechanical properties andbiostability of the implanted device. Preferably, the fibrinogen andthrombin used to make fibrin in the present invention are from the sameanimal or human species as that in which the stent will be implanted inorder to avoid any inter-species immune reactions, e.g., human anti-cow.The fibrin product can be in the form of a fine, fibrin film produced bycasting the combined fibrinogen and thrombin in a film and then removingmoisture from the film osmotically through a semipermeable membrane. Inthe European Pat. Application 0366564, a substrate (preferably havinghigh porosity or high affinity for either thrombin or fibrinogen) iscontacted with a fibrinogen solution and with a thrombin solution. Theresult is a fibrin layer formed by polymerization of fibrinogen on thesurface of the medical device. Multiple layers of fibrin applied by thismethod could provide a fibrin layer of any desired thickness.Alternatively, the fibrin can first be clotted and then ground into apowder which is mixed with water and stamped into a desired shape in aheated mold (U.S. Pat. No. 3,523,807). Increased stability can also beachieved in the shaped fibrin by contacting the fibrin with a fixingagent such as glutaraldehyde or formaldehyde. These and other methodsknown by those skilled in the art for making and forming fibrin may beused in the present invention.

If a synthetic graft is coated with collagen, the methods for preparingcollagen and forming it on synthetic graft devices are well known as setforth in U.S. Pat. No. 5,851,230 to Weadock et al., incorporated hereinby reference. This patent describes methods for coating a syntheticgraft with collagen. Methods for adhering collagen to a porous graftsubstrate typically include applying a collagen dispersion to thesubstrate, allowing it to dry and repeating the process. Collagendispersions are typically made by blending insoluble collagen(approximately 1-2% by weight) in a dispersion at acidic pH (a pH in arange of 2 to 4). The dispersion is typically injected via syringe intothe lumen of a graft and massaged manually to cover the entire innersurface area with the collagen slurry. Excess collagen slurry is removedthrough one of the open ends of the graft. Coating and drying steps arerepeated several times to provide sufficient treatment.

In yet another embodiment, the stent or synthetic graft is coated withamorphous carbon. In U.S. Pat. No. 5,198,263, incorporated herein byreference, a method for producing a high-rate, low-temperaturedeposition of amorphous carbon films in the presence of a fluorinated orother halide gas is described. Deposition according to the methods ofthis invention can be performed at less than 100° C., including ambientroom temperature, with a radio-frequency, plasma-assisted,chemical-vapor deposition process. The amorphous carbon film producedusing the methods of this invention adheres well to many types ofsubstrates, including for example glasses, metals, semiconductors, andplastics.

Attachment of a fullerene moiety to reactive amino group sites of anamine-containing polymer to form the fullerene-graft, amine-containingpolymers may be performed as described in U.S. Pat. No. 5,292,813.Chemical modification in this manner allows for direct incorporation ofthe fullerenes into the stent. In another embodiment, the fullerenes maybe deposited on the surface of the stent or synthetic grafts asdescribed above. (see, WO 99/32184 to Leone et al., incorporated byreference). Fullerenes (e.g., C₆₀) may also be attached through anepoxide bond to the surface of stainless steel (Yamago et al., ChemicalDerivatization of Organofullerenes through Oxidation, Reduction and C—Oand C—C Bond Forming Reactions. J. Org. Chem., 58 4796-4798 (1998),incorporated herein by reference). The attachment is through a covalentlinkage to the oxygen. This compound and the protocols for coupling arecommercially available from BuckyUSA. (BuckyUSA, Houston, Tex.).

(E) Addition of Antibodies and growth factor to the Matrix—Antibodiesthat promote adherence of progenitor endothelial cells, and growthfactors for promoting cell growth and differentiation are incorporatedinto the matrix, either covalently or noncovalently. Antibodies andgrowth factor may be incorporated into the matrix layer by mixing theantibodies and growth factor with the matrix coating solution and thenapplied to the surface of the device. Usually, antibodies and growthfactors are attached to the surface of the outermost layer of matrixthat is applied on the luminal surface of the device, so that theantibodies and growth factor are projecting on the surface that is incontact with the circulating blood. Antibodies and growth factors areapplied to the surface matrix using standard techniques.

In one embodiment, the antibodies are added to a solution containing thematrix. For example, Fab fragments on anti-CD34 monoclonal antibody areincubated with a solution containing human fibrinogen at a concentrationof between 500 and 800 mg/dl. It will be appreciated that theconcentration of anti-CD34 Fab fragment will vary and that one ofordinary skill in the art could determine the optimal concentrationwithout undue experimentation. The stent is added to the Fab/fibrinmixture and the fibrin activated by addition of concentrated thrombin(at a concentration of at least 1000 U/ml). The resulting polymerizedfibrin mixture containing the Fab fragments incorporated directly intothe matrix is pressed into a thin film (less than 100 μm) on the surfaceof the stent or synthetic graft. Virtually any type of antibody orantibody fragment can be incorporated in this manner into a matrixsolution prior to coating of a stent or synthetic graft.

For example, in another embodiment, whole antibodies with or withoutantibody fragments and growth factors are covalently coupled to thematrix. In one embodiment, the antibodies and growth factor(s) aretethered covalently the matrix through the use of hetero- orhomobifunctional linker molecules. As used herein the term “tethered”refers to a covalent coupling of the antibody to the matrix by a linkermolecule. The use of linker molecules in connection with the presentinvention typically involves covalently coupling the linker molecules tothe matrix after it is adhered to the stent. After covalent coupling tothe matrix, the linker molecules provide the matrix with a number offunctionally active groups that can be used to covalently couple one ormore types of antibody. FIG. 1A provides an illustration of coupling viaa cross-linking molecule. An endothelial cell, 1.01, binds to anantibody, 1.03, by a cell surface antigen, 1.02. The antibody istethered to the matrix, 1.05-1.06, by a cross-linking molecule, 1.04.The matrix, 1.05-1.06, adheres to the stent, 1.07. The linker moleculesmay be coupled to the matrix directly (i.e., through the carboxylgroups), or through well-known coupling chemistries, such as,esterification, amidation, and acylation. The linker molecule may be adi- or tri-amine functional compound that is coupled to the matrixthrough the direct formation of amide bonds, and providesamine-functional groups that are available for reaction with theantibodies. For example, the linker molecule could be a polyaminefunctional polymer such as polyethyleneimine (PEI), polyallylamine(PALLA) or polyethyleneglycol (PEG). A variety of PEG derivatives, e.g.,mPEG-succinimidyl propionate or mPEG-N-hydroxysuccinimide, together withprotocols for covalent coupling, are commercially available fromShearwater Corporation, Birmingham, Ala. (See also, Weiner et al.,Influence of a poly-ethyleneglycol spacer on antigen capture byimmobilized antibodies. J. Biochem. Biophys. Methods 45:211-219 (2000),incorporated herein by reference). It will be appreciated that theselection of the particular coupling agent may depend on the type ofantibody used and that such selection may be made without undueexperimentation. Mixtures of these polymers can also be used. Thesemolecules contain a plurality of pendant amine-functional groups thatcan be used to surface-immobilize one or more antibodies.

Antibodies may be attached to C₆₀ fullerene layers that have beendeposited directly on the surface of the stent. Cross linking agents maybe covalently attached to the fullerenes. The antibodies are thenattached to the cross-linking agent, which in turn is attached to thestent. FIG. 1B provides an illustration of coupling by C₆₀. Theendothelial cell, 2.01, is bound via a cell surface antigen, 2.02, to anantibody, 2.03, which in turn is bound, covalently or non-covalently tothe matrix, 2.04. The matrix, 2.04, is coupled covalently via C₆₀, 2.05,to the stent, 2.06.

Small molecules of the invention comprise synthetic or naturallyoccurring molecules or peptides which can be used in place ofantibodies, growth factors or fragments thereof. For example, lectin isa sugar-binding peptide of non-immune origin which occurs naturally. Theendothelial cell specific Lectin antigen (Ulex Europaeus Uea 1) (Schatzet al. 2000 Human Endometrial Endothelial Cells: Isolation,Characterization, and Inflammatory-Mediated Expression of Tissue Factorand Type 1 Plasminogen Activator Inhibitor. Biol Reprod 62: 691-697) canselectively bind the cell surface of progenitor endothelial cells.

Synthetic “small molecules” have been created to target various cellsurface , proteins, glucoproteins, polysaccharides and receptors. Thesemolecules selectively bind a specific surface moieties and can targetspecific cell types such as progenitor endothelial cells. Smallmolecules can be synthesized to recognize endothelial cell surfacemarkers such as VEGF. SU11248 (Sugen Inc.) (Mendel et al. 2003 In vivoantitumor activity of SU11248, a novel tyrosine kinase inhibitortargeting vascular endothelial growth factor and platelet-derived growthfactor receptors: determination of a pharmacokinetic/pharmacodynamicrelationship. Clin Cancer Res. January;9(1):327-37), PTK787/ZK222584(Drevs J. et al. 2003 Receptor tyrosine kinases: the main targets fornew anticancer therapy. Curr Druq Targets. February;4(2):113-21) andSU6668 (Laird, AD et al. 2002 SU6668 inhibits Flk-1/KDR and PDGFRbeta invivo, resulting in rapid apoptosis of tumor vasculature and tumorregression in mice. FASEB J. May;16(7):681-90) are small molecules whichbind to VEGFR-2.

Another subset of synthetic small molecules which target the endothelialcell surface are the alpha(v)beta(3) integrin inhibitors. SM256 andSD983 (Kerr J S. et al. 1999 Novel small molecule alpha v integrinantagonists: comparative anti-cancer efficacy with known angiogenesisinhibitors. Anticancer Res March-April;19(2A):959-68) are both syntheticmolecules which target and bind to alpha(v)beta(3) present on thesurface of endothelial cells.

The present invention provides a drug delivery system comprising: coatedmedical devices such as stents, stent grafts, heart valves, catheters,vascular prosthetic filters, artificial heart, external and internalleft ventricular assist devices (LVADs), and synthetic vascular grafts,for the treatment of diseases, including tumor and vascular diseases,such as restenosis, artherosclerosis, thrombosis, blood vesselobstruction, and the like. In one embodiment, the coating on the presentmedical device comprises a biocompatible matrix, at least one antibody,and at least one compound such as a ligand.

Transgenic cells incorporating at least one transgene that is introducedinto the cells by viral or non-viral based genetic procedures. Thetransgene codes for at least one therapeutic drug and is expressedcontinuously or upon induction. In one embodiment, the therapeutic drugis a protein. The transgenic cells also present at least one antigen onits cell surface that can be recognized and bound by the antibody thatis coated on the surface of the medical device.

As used herein “antibody” refers to antibody or antibody fragment, or acombination of antibody and fragments, which can be a monoclonalantibody, a polyclonal antibody, a chimeric antibody, or a humanizedantibody. The antibody fragment of the invention comprises any fragmentsize, such as large and small molecules which retain the characteristicto recognize and bind the target antigen as the antibody (FIGS. 1A, 1B,and 11).

As used herein “ligand” refers to a molecule that binds a receptor onthe mammalian cell. For example, the ligand can be an antibody, antibodyfragment (FIGS. 1A, 1B, 11, and 17), cell adhesion molecule, basementmembrane component which recognizes and binds a specific epitope orstructure on the membrane of the cell. In the embodiment of theinvention which uses genetically altered cells, the ligand can bespecifically selected to recognize and bind to a gene product producedby the exogenous DNA introduced into the cells.

As used herein “protein” refers to a polymer of amino acids of anylength. The polymer may be linear or branched, may comprise modifiedamino acids, and may be interrupted by non-amino acids. The polymer maybe naturally occurring peptides, proteins, or modified and syntheticforms thereof including biologically active fragments, derivatives,analogues, mimetics, and non-functional or dominant negative mutants.

The medical device of this invention can be any device used forimplanting into an organ or body part comprising a lumen, and can be,but is not limited to, a stent, a stent graft, a synthetic vasculargraft, a heart valve, a catheter, a vascular prosthetic filter, apacemaker, a pacemaker lead, a defibrilator, a patent foramen ovale(PFO) septal closure device, a vascular clip, a vascular aneurysmoccluder, a hemodialysis graft, a hemodialysis catheter, anatrioventricular shunt, an aortic aneurysm graft device or components, avenous valve, a suture, a vascular anastomosis clip, an indwellingvenous or arterial catheter, a vascular sheath and a drug delivery port.The medical device can be made of numerous materials depending on thedevice. For example, a stent of the invention can be made of stainlesssteel, Nitinol (NiTi), or chromium alloy. Synthetic vascular grafts canbe made of a cross-linked PVA hydrogel, polytetrafluoroethylene (PTFE),expanded polytetrafluoroethylene (ePTFE), porous high densitypolyethylene (HDPE), polyurethane, and polyethylene terephthalate.

The biocompatible matrix forming the coating of the present devicecomprises a synthetic material such as polyurethanes, segmentedpolyurethane-urea/heparin, poly-L-lactic acid, cellulose ester,polyethylene glycol, polyvinyl acetate, dextran and gelatin, anaturally-occurring material such as basement membrane components suchas collagen, elastin, laminin, fibronectin, vitronectin; heparin,fibrin, cellulose, and amorphous carbon, or fullerenes.

In one embodiment, the medical device comprises a biocompatible matrixcomprising fullerenes. In this embodiment, the fullerene can range fromabout C₂₀ to about C₁₅₀ in the number of carbon atoms, and moreparticularly, the fullerene is C₆₀ or C₇₀. The fullerene of theinvention can also be arranged as nanotubes on the surface of themedical device.

The antibody for providing to the coating of the medical devicecomprises at least one antibody that recognizes and binds a transgeniccell surface antigen which can be expressed by an endogenous gene or bya transgene and modulates the adherence of the cells onto the surface ofthe medical device. The antibody can be covalently or noncovalentlyattached to the surface of the matrix, or tethered covalently by alinker molecule to the outermost layer of the matrix coating the medicaldevice. In this aspect of the invention, for example, the monoclonalantibodies can further comprises Fab or F (ab′) 2 fragments.

The antibody of the invention recognizes and binds antigens withspecificity for the mammal being treated and their specificity is notdependent on cell lineage. In one embodiment, the antibody is specificfor a human progenitor endothelial cell surface antigen such as CD133,CD14, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146),CD130, stem cell antigen (Sca-1), stem cell factor 1(SCF/c-Kit ligand),Tie-2, HAD-DR and others.

In another embodiment, the coating of the medical device comprises atleast one layer of a biocompatible matrix as described above, the matrixcomprising an outer surface for attaching a therapeutically effectiveamount of at least one type of small molecule of natural or syntheticorigin. The small molecule recognizes and interacts with an antigen on atransgenic cell surface to immobilize the transgenic cell on the surfaceof the device and to induce transgene expression. The small moleculescan be derived from a variety of sources such as cellular componentssuch as fatty acids, proteins, nucleic acids, saccharides and the likeand can interact with a receptor on the surface of a transgenic cell. Inthis embodiment of the invention, the coating on the medical device canfurther comprise a compound such as a ligand in conjunction with thecoating comprising an antibody.

Both viral and non-viral based genetic procedures can be used tointroduce transgenes for generating transgenic cells. Transgenic cellsof the invention express and secrete therapeutic drugs coded bytransgenes that are either transiently or stably incorporated.Additional transgenes can be incorporated to confer survival, selectionand/or growth advantage. Various cells such as endothelial cells orleukocytes including neutrophil, eosinophil, basophil, monocyte andlymphocytes or somatic cells, or a combination of these cells can bemodified to produce transgenic cells, which may be eithernon-repopulating or repopulating. Transgenic cells can be cultured invitro, collected, and stored. Transgenic cells producing a variety oftherapeutic drugs can be generated by incorporating different transgenesto serve different therapeutic purposes. Transgenic cells can beadministered as a single or mixed populations via systemic or localroutes. Various amounts of transgenic cells can be administered torelease different amount of therapeutic drugs upon individualconditions. In one embodiment, transgenic cells are repopulatingprogenitor endothelial cells. In a further embodiment, transgenicprogenitor endothelial cells are administered locally with catheterbased delivery or dual balloon inflation method.

In one embodiment, transgenic cells further comprise an additionaltransgene that expresses an exogenous cell surface antigen, which can bespecifically recognized and bound by the antibody that is coated in thematrix of the medical device. Transgene expression and product secretioncan be continuous or contingent upon the activation of an induciblepromoter via exogenous excitation.

The therapeutic compounds coded by the transgenes of the invention canbe any molecule with a desired physiological effect, and can be, but isnot limited to, proteins as defined including growth factors, chemokinesand cytokines, ligands and receptors, and other functional proteins andnon-protein excretable compounds. In one embodiment, a therapeuticcompound is a protein selected from the group consisting of endothelialnitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF),an anti-inflammatory factor, and an inflammation-modulating factor.

The exciting drug for transgene expression and product secretion of theinvention can be a ligand that binds the transgenic cell surface antigenand triggers downstream signaling pathway activation, or can be taken upby the transgenic cell and stimulate gene expression through aninducible promoter. In one embodiment, the ligand or drug isadministered systemically. In another embodiment, the ligand or drug iscoated in the matrix of the implanted device and administered locally.

The invention provides methods for treating a variety of diseases, whichcan be, but not limited to, tumors, vascular diseases, and healingresponse. The methods provide improvement over prior art in terms oftarget site delivery of a variety of drugs of desired amount upondemand.

The invention provides a method for treating tumors and theirmetastases. In this embodiment, the transgene can code for (1) anantiangiogenic factor, such as interferons (IFNs), thrombospondin (TSP),angiostatin, endostatin, oncostatin M (OSM), and Rho, which inhibitsneovascularization that is a prerequisite for tumor progressive growth;or (2) an tumor suppressive protein, such as p53, Rb, E1, BRCA1,antibody or dominant negative mutant of a cell growth activator such asa growth factor, a cyclin dependent kinase (CDK) or a cyclin, E2F, NFκB;or a combination of these genes.

As used herein the phrase “anti-angiogenic factor” refers to a moleculethat is capable of inhibiting angiogenesis.

The invention also provides methods for treating vascular disease. Inone embodiment, the invention is used to treat ischemic conditions, inwhich the transgene codes for an angiogenic factor such as pleiotrophin,angiogenin, angiopoietin, an integrin stimulating factor, or an antibodyor dominant negative mutant of an anti-angiogenic factor.

As used herein the phrase “angiogenic factor” refers to a molecule thatis capable of stimulating angiogenesis.

In another embodiment, the invention is used to treat atherosclerosis,restenosis, thrombosis, aneurysm or blood vessel obstruction. In thisembodiment of the invention, transgene can code for (a) eNOS or VEGFthat promotes re-endothelialization; or (b) an anti-inflammatory orinflammation-modulating factor such as IFN-β, IFN-α, TGF-β, orinterleukin-10 (IL-10); or (c) an inhibitor of smooth muscle cellgrowth, migration, or differentiation that inhibits intimal hyperplasia;or a combination of these genes.

The invention also provides an engineered method for inducing a healingresponse. In one embodiment, a method is provided for rapidly inducingthe formation of a confluent layer of endothelium in the luminal surfaceof an implanted device in a target lesion of an implanted vessel, inwhich transgenic cells are progenitor endothelial cells that expresseNOS, VEGF, or an anti-inflammatory or inflammation-modulating factor.In this embodiment of the invention, a medical device is provided ofincreased biocompatibility over prior art devices, and decreases orinhibits tissue-based excessive intimal hyperplasia and restenosis bydecreasing or inhibiting smooth muscle cell migration, smooth musclecell differentiation, and collagen deposition along the inner luminalsurface at the site of implantation of the medical device.

In one embodiment of the invention, a method for coating a medicaldevice comprises the steps of: applying at least one layer of abiocompatible matrix to the surface of the medical device, wherein thebiocompatible matrix comprises at least one component selected from thegroup consisting of a polyurethane, a segmentedpolyurethane-urea/heparin, a poly-L-lactic acid, a cellulose ester, apolyethylene glycol, a polyvinyl acetate, a dextran, gelatin, collagen,elastin, laminin, fibronectin, vitronectin, heparin, fibrin, celluloseand carbon and fullerene, and applying to the biocompatible matrix,simultaneously or sequentially, at least one antibody, and optionallyone compound which induces transgene expression.

The invention further provides a method for treating diseases such astumor, vascular disease, and wound healing in a mammal comprisesimplanting a medical device into a vessel or tubular organ of themammal, wherein the medical device is coated with (a) a biocompatiblematrix; (b) at least one antibody; and optionally (c) one compound,introducing transgenic cells into the mammal that is need of thetreatment, and optionally administering a compound, wherein the antibodycoated in the matrix of the medical device recognizes and binds anantigen expressed on the transgenic cell surface, so that the transgeniccells are immobilized on the surface of the matrix, and at least onetherapeutic drug coded by a transgene is expressed upon the compoundexcitation and secreted at a designated site.

The invention further provides a method for treating vascular disease ina mammal comprises implanting a medical device into a vessel or tubularorgan of the mammal, wherein the medical device is coated with (a) abiocompatible matrix, (b) at least one antibody, and optionally (c) onecompound, and introducing transgenic cells into the mammal that is inneed of the treatment, and optionally administering a compound, whereinthe antibody coated in the matrix of the medical device recognizes andbinds an antigen expressed only on the transgenic cell surface so thatthe transgenic cells are immobilized on the surface of the matrix. Thetransgenic (genetically-altered) cells contain genetic material whichencodes at least one therapeutic drug constitutively or upon activationby a signal such as a compound.

The present transgenic cells contain at least one expressible transgenethat can code for, but not limited to (1) growth factors includingfamily members such as platelet derived growth factor (PDGF),transforming growth factor (TGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), insulin like growth factors (IGF),vascular endothelial growth factor (VEGF), heparin binding growthfactors, hepatoma-derived growth factor (HDGF), hepatocyte growthfactor/scatter factor (HGF), placental growth factor (PIGF), plateletderived endothelial cell growth factor (PD-ECGF), stem cell factor(SCF), and their other protein forms; (2) Chemokines such as CXCfamilies, CC families, C families, and their other protein forms; (3)cytokines such as a disintegrin and metalloprotease (ADAM), annexin V,B7 & CD28/CTLA-4 receptor families, bone morphogenetic protein (BMP),caspase, CD44, CD44H, endothelin-1 (ET-1), eph, erythropoietin (Epo),intercellular adhesion molecule-3/CD50 (ICAM-3), macrophage stimulatingprotein (MSP), matrix metalloproteinase (MMP), neurotrophic factors,endothelial nitric oxide synthase (eNOS), NKG2D, platelet endothelialcell adhesion molecule-1 (PECAM-1/CD31), pleiotrophin/midkine (PTN/MK),transferrin receptor (sTfR), hedgehog, STAT, stem cell marker, Th1/Th2,thrombopoietin (Tpo), tumor necrosis factor family, VCAM-1/CD16,monoclonal non-specific suppressor factor beta (MNSFbeta), 6Ckine (SLC),B-lymphocyte chemoattractant (BCA-1/BLC), leukemia inhibitory factor,monocyte-derived neutrophil-activating peptide (GRO), and their otherprotein forms; (4) other functional proteins invovled in the regulationof signal transduction, cell cycle regulation, cell division, and/orcell differentiation, such as ligands, receptors, phosphorylases,kinases, transcriptional factors, and their other protein forms.

In one embodiment, antiangiogenic factors for use in the invention are,for example, interferons (IFNs), thrombospondin (TSP), angiostatin, andendostatin, oncostatin M (OSM), blockers of integrin engagement,metalloproteinases inhibitors, inhibitors of endothelial cellphosphorylation, dominant negative receptors for angiogenesis inducers,antibodies of angiogenesis inducers, other proteins acting by othermeans, and their other protein forms. Other angiogenic factors includeangiogenin, angiopoietins, integrin stimulating factors such as Del-1,and their other protein forms.

Additional growth factors for use in the invention are, for example,pleiotrophin, midkines, VEGF family including VEGF-2, VEGF-C, andVEGF-D, FGF family, including FGF-1, FGF-2, FGF-5, and FGF-18,hepatoma-derived growth factor (HDGF), hepatocyte growth factor/scatterfactor (HGF), members of the epidermal growth factor (EGF) family,including transforming growth factor alpha, EGF, and TGF-alpha-HIII, andplatelet derived growth factor (PDGF), including AA, AB, and BBisoforms.

EXPERIMENTAL EXAMPLES

This invention is illustrated in the experimental details section whichfollows. These sections set forth below the understanding of theinvention, but are not intended to, and should not be construed to,limit in any way the invention as set forth in the claims which followthereafter.

Example 1

Endothelial Progenitor Cell Phenotyping

Endothelial Progenitor Cells (EPC) were isolated either by CD34+MagneticBead Isolation (Dynal Biotech) or enriched medium isolation as describedrecently (Asahara T, Murohara T, Sullivan A, et al. Isolation ofputative progenitor endothelial cells for angiogenesis. Science1997;275:964-7). Briefly, peripheral venous blood was taken from healthymale volunteers and the mononuclear cell fraction was isolated bydensity gradient centrifugation, and the cells were plated on humanfibronectin coated culture slides (Becton Dickinson) in EC basalmedium-2 (EBM-2) (Clonetics) supplemented with 5% fetal bovine serum,human VEGF-A, human fibroblast growth factor-2, human epidermal growthfactor, insulin-like growth factor-1, and ascorbic acid. EPCs were grownup to seven days with culture media changes every 48 hours. The resultsof these experiments are shown in FIGS. 2A and 2B. FIGS. 2A and 2B showthat the anti-CD34 isolated cell appear more spindle-like, whichindicates that the cells are differentiating into endothelial cells.

EC phenotype was determined by immunohistochemistry. Briefly, EPC werefixed in 2% Paraformaldehyde (PFA) (Sigma) in Phosphate buffered saline(PBS) (Sigma) for 10 minutes, washed 3× with PBS and stained withvarious EC specific markers; rabbit anti-human VEGFR-2 (AlphaDiagnostics Intl. Inc.), mouse anti-human Tie-2 (Clone Ab33, UpstateBiotechnology), mouse anti-human CD34 (Becton Dickinson), EC-Lectin(Ulex Europaeus Uea 1) (Sigma) and mouse anti-human Factor 8 (Sigma).The presence of antibody was confirmed by exposure of the cells to afluorescein isothiocyanate-conjugated (FITC) secondary antibody.Propidium Iodine (PI) was used as a nuclear marker. The results of theseexperiments are shown in FIGS. 2C-2G. FIG. 2C shows that VEGFR-2 isexpressed after 24 hours in culture, confirming that the cells areendothelial cells. FIGS. 2D and 2F show the nuclear staining of thebound cells after 7 days of incubation and FIGS. 2E and 2G the samefield of cells stained with an FITC conjugated anti-Tie-2 antibody.

EPCs ability to express endothelial nitric oxide synthase (eNOS), ahallmark of EC function, was determined by ReverseTranscriptase-Polymerase Chain Reaction (rt-PCR) for eNOS mRNA. EPCswere grown up to seven days in EBM-2 medium after which total RNA wasisolated using the GenElute Mammalian total RNA kit (Sigma) andquantified by absorbance at 260 nm. Total RNA was reverse transcribed in20 μL volumes using Omniscript RT kit (Qiagen) with 1 μg of randomprimers. For each RT product, aliquots (2-10 μL) of the final reactionvolume were amplified in two parallel PCR reactions using eNOS (299 bpproduct, sense 5′-TTCCGGGGATTCTGGCAGGAG-3′, antisense5′-GCCATGGTMCATCGCCGCAG-3′) or GAPDH (343 bp product, sense5′-CTCTMGGCTGTGGGCAAGGTCAT-3′, antisense 5′-GAGATCCACCACCCTGTTGCTGTA-3′)specific primers and Taq polymerase (Pharmacia Biotech Amersham). PCRcycles were as follows: 94° C. for 5 minutes, 65° C. for 45 seconds, 72°C. for 30 seconds (35 cycles for eNOS and 25 cycles for GAPDH). rt-PCRproducts were analyzed by 2% agarose gel electrophoresis, visualizedusing ethidium bromide and quantified by densitometry. The results ofthis experiment are shown in FIGS. 3A and 3B. As seen in FIGS. 3A and3B, nitric oxide synthetase (eNOS) is expressed after the cells havebeen incubated in medium for 3 days in culture in the presence orabsence of oxygen. eNOS mRNA expression continues to be present after7-days in culture. The presence of eNOS mRNA indicates that the cellshave differentiated into mature endothelial cells by day 3 and havebegun to function like fully differentiated endothelial cells.

Example 2

Endothelial Cell Capture by anti-CD34 coated Stainless Steel Disks:Human Umbilical Vein Endothelial Cells (HUVEC) (American Type CultureCollection) are grown in endothelial cell growth medium for the durationof the experiments. Cells are incubated with CMDX and gelatin coatedsamples with or without bound antibody on their surface or barestainless steel (SST) samples. After incubation, the growth medium isremoved and the samples are washed twice in PBS. Cells are fixed in 2%paraformaldehyde (PFA) for 10 minutes and washed three times, 10 minuteseach wash, in PBS, to ensure all the fixing agent is removed. Eachsample is incubated with blocking solution for 30 minutes at roomtemperature, to block all non-specific binding. The samples are washedonce with PBS and the exposed to 1:100 dilution of VEGFR-2 antibody andincubated overnight. The samples are subsequently washed three timeswith PBS to ensure all primary antibody has been removed.FITC-conjugated secondary antibody in blocking solution is added to eachrespective sample at a dilution of 1:100 and incubated for 45 minutes atroom temperature on a Belly Dancer apparatus. After incubation, thesamples are washed three times in PBS, once with PBS containing 0.1%Tween 20, and then again in PBS. The samples are mounted with PropidiumIodine (PI) and visualized under confocal microscopy.

FIGS. 4A-4E are photomicrographs of SST samples coated with CMDX andanti-CD34 antibody (FIG. 4A), gelatin and anti-CD34 antibody coated(FIG. 4B), bare SST (FIG. 4C), CMDX coated and no antibody (FIG, 4D) andgelatin-coated and no antibody (FIG. 4E). The figures show that only theantibody coated samples contain numerous cells attached to the surfaceof the sample as shown by PI staining. The bare SST control disk showsfew cells attached to its surface.

FIGS. 5A-5C are photomicrographs of control samples CMDX-coated withoutantibody bound to its surface. FIG. 5A shows very few cells as seen byPI staining adhered to the surface of the sample. FIG. 5B shows that theadherent cells are VEGFR-2 positive indicating that they are endothelialcells and FIG. 5C shows a combination of the stained nuclei and theVEGFR-2 positive green fluorescence. FIGS. 5D-F are photomicrographs ofcontrol samples coated with gelatin without antibody on its surface.FIG. 5D shows no cells are present since PI staining is not present inthe sample and there is no green fluorescence emitted by the samples(see FIGS. 5E and 5F).

FIGS. 6A-6C are photomicrographs of CMDX coated SST samples havinganti-CD34 antibody bound on its surface. The figures show that thesamples contain numerous adherent cells which have established a nearconfluent monolayer (FIG. 6A) and which are VEGFR-2 positive (FIGS. 6Band 6C) as shown by the green fluorescence. Similarly, FIGS. 6D-6F arephotomicrographs of a gelatin-coated sample with anti-CD34 antibodybound to its surface. These figures also show that HUVECs attached tothe surface of the sample as shown by the numerous red-stained nucleiand green fluorescence from the VEGFR-2/FITC antibody (FIGS. 6E and 6F).

Example 3

VEGFR-2 and Tie-2 Staining of Progenitor Endothelial Cells: Progenitorcell are isolated from human blood as described in the in Example 1 andincubated in growth medium for 24 hours, 7 days, and 3 weeks in vitro.After incubation, the growth medium is removed and the samples arewashed twice in PBS. Cells are fixed in 2% paraformaldehyde (PFA) for 10minutes and washed three times, 10 minutes each wash, in PBS, to ensureall the fixing agent is removed. Each sample is incubated with 440 μl ofGoat (for VEGFR-2) or Horse (for Tie-2) blocking solution for 30 minutesat room temperature, to block all non-specific binding. The samples arewashed once with PBS and the VEGFR-2 or Tie-2 antibody was added at adilution of 1:100 in blocking solution and the samples are incubatedovernight. The samples are then washed three times with PBS to ensureall primary antibody has been washed away. FITC-conjugated secondaryantibody (200 μl) in horse or goat blocking solution is added to eachrespective sample at a dilution of 1:100 and incubated for 45 minutes atroom temperature on a Belly Dancer apparatus. After incubation, thesamples are washed three times in PBS, once with PBS containing 0.1%Tween 20, and then again in PBS. The samples are mounted with PropidiumIodine (PI) and visualized under confocal microscopy.

FIG. 7 is a photomicrograph of a CMDX-coated sample containing CD34antibody on its surface which was incubated with the cells for 24 hours,and shows that progenitor cells were captured on the surface of thesample and as demonstrated by the red-stained nuclei present on thesurface of the sample. The figure also shows that about 75% of the cellsare VEGFR-2 positive with a round morphology.

FIGS. 8A and 8B are from a sample which was incubated with the cells for7 days. As seen in FIG. 8A, there are cells present on the sample asshown by the red-stained nuclei, which are VEGFR-2 positive (FIG. 8B,100%) and are more endothelial in structure as shown by the spindleshape of the cells. FIGS. 9A and 9B are photomicrographs of CMDX-coatedsample containing CD34 antibody on its surface, which was incubated for7 days with the cells and after incubation, the sample was exposed toTie-2 antibody. As seen in FIG. 9A, there are numerous cells attached tothe surface of the samples as shown by the red-stained nuclei. The cellsadhered to the sample are also Tie-2 positive (100%) as seen by thegreen fluorescence emitted from the cells (FIG. 9B). In summary, after 7days of incubation of the cells with the samples, the CD34antibody-coated samples are able to capture endothelial cells on theirsurface as seen by the numerous cells attached to the surface of thesamples and the presence of VEGFR-2 and Tie-2 receptors on the surfaceof the adhered cells. In addition, the presence of 100% endothelialcells on the surface of the samples at 7 days indicates that thenon-endothelial cells may have detached or that all adherent cells havebegun to express endothelial cell markers by day 7.

FIGS. 10A-10C are phase contrast photomicrographs of the progenitorendothelial cells grown for 3 weeks in endothelial cell growth medium.FIG. 10A demonstrates the cells have differentiated into maturedendothelial cells as shown by the two-dimensional tube-like structures(arrow) reminiscent of a lumen of a blood vessel at the arrow. FIG. 10Bshows that,there is a three-dimensional build-up of cells in multiplelayers; i.e.; one on top of the other, which confirms reports thatendothelial cells grown for prolonged periods of time begin to formlayers one on top of the other. FIG. 10C shows progenitor cells growingin culture 3 weeks after plating which have the appearance ofendothelial cells, and the figure confirms that the cells areendothelial cells as demonstrated by the green fluorescence of theCD34/FITC antibodies present on their surface.

The above data demonstrate that white blood cells isolated from humanblood have CD34 positive progenitor cells and that these cells candevelop into mature endothelial cells and readily express endothelialcell surface antigens. (VEGFR-2 and Tie-2) The data also show thatantibodies against progenitor or stem cell surface antigens can be usedto capture these cells on the surface of a coated medical device of theinvention.

Example 4 Fullerene Coated and Fullerene Coated with Anti-CD34 Antibodyand/or an Endothelial Cell Growth Factor (Ang-2, VEGF) Stainless Steel

Stainless steel stents and disks are derivatized with a functionalfullerene layer for attaching antibodies and/or growth factors (i.e.,VEGF or Ang-2) using the following procedure:

In the first step, the surface of the SST stent or disk is activatedwith 0.5M HCL which also cleans the surface of any passivatingcontaminants. The metal samples are removed from the activation bath,rinsed with distilled water, dried with methanol and oven-dried at 75°C. The stents are then immersed in the toluene derivative solution withfullerene oxide (C₆₀-O), for a period of up to 24 hours. The fullereneoxide binds to the stent via Fe—O, Cr—O and Ni—O found on the stent. Thestents are removed from the derivatizing bath, rinsed with toluene, andplaced in a Soxhlet Extractor for 16 hours with fresh toluene to removeany physisorbed C₆₀. The stents are removed and oven-dried at 105° C.overnight. This reaction yields a fully derivatized stent or disk with amonolayer of fullerenes.

In step 2 a di-aldehyde molecule is formed in solution by reactingsebacic acid with thionyl chloride or sulfur oxychloride (SOCl₂) to formSebacoyl chloride. The resultant Sebacoyl chloride is reacted withLiAl[t-OButyl]₃ H and diglyme to yield 1,10-decanediol as shown below:

In step 3, an N-methyl pyrolidine derivate is formed on the surface ofthe stent or disk (from step 1). The fullerene molecule is furtherderivatized by reacting equimolar amounts of fullerene andN-methylglycine with the 1,10-decanediol product of the reaction of step2, in refluxing toluene solution under nitrogen for 48 hours to yieldN-methyl pyrolidine-derivatized fullerene-stainless steel stent or diskas depicted below.

The derivatized stainless steel stent or disk is washed to remove anychemical residue and used to bind the antibodies and/or (VEGF or Ang-2)using standard procedures. Progenitor cell are isolated from human bloodas described in Example 1 and exposed to the anti-CD34 antibody coatedfullerene disks. After incubation, the growth medium is removed and thesamples are washed twice in PBS. Cells are fixed in 2% paraformaldehyde(PFA) for 10 minutes and washed three times, 10 minutes each wash, inPBS, to ensure all the fixing agent is removed. Each sample is incubatedwith blocking solution for 30 minutes at room temperature, to block allnon-specific binding. The samples are washed once with PBS and theexposed to 1:100 dilution of VEGFR-2 antibody and incubated overnight.The samples are subsequently washed three times with PBS to ensure allprimary antibody has been removed. FITC-conjugated secondary antibody inblocking solution is added to each respective sample at a dilution of1:100 and incubated for 45 minutes at room temperature on a Belly Dancerapparatus. After incubation, the samples are washed three times in PBS,once with PBS containing 0.1% Tween 20, and then again in PBS. Thesamples are mounted with Propidium Iodine (PI) and visualized underconfocal microscopy. FIG. 11 shows a schematic representation of afunctional fullerene coated stent surface of the invention binding aprogenitor cell. FIGS. 12A-12B are, respectively, photomicrographs offullerene-coated control sample without antibody stained with PI (12A)and anti-VEGFR-2/FITC-conjugated antibody stained. FIGS. 12C and 12D arephotomicrographs of a sample coated with a fullerene/anti-CD34 antibodycoating. As shown in the figures, the anti-CD34 antibody coated samplecontains more cells attached to the surface which are VEGFR-2 positive.

Fullerene-coated samples with and without antibodies are implanted intoYorkshire pigs as described in Example 5. The stents are explanted forhistology and the stented segments are flushed with 10% bufferedFormalin for 30 seconds followed by fixation with 10% buffered Formalinuntil processed. Five sections are cut from each stent; 1 mm proximal tothe stent, 1 mm from the proximal end of the stent, mid stent, 1 mm fromthe distal edge of the stent and 1 mm distal to the stent. Sections arestained with Hematoxylin & Eosin (HE) and Elastin Trichrome. FIGS.13A-13D are photomicrographs of cross-sections through coronary arteryexplants of stents which had been implanted for 4 weeks. The data showthat the fullerene-coated (FIGS. 13B and 13D) stents inhibit excessiveintimal hyperplasia at the stent site over the control (bare stent,FIGS. 13A and 13C).

Example 5

PORCINE BALLOON INJURY STUDIES: Implantation of antibody-covered stentsis performed in juvenile Yorkshire pigs weighing between 25 and 30 kg.Animal care complies with the “Guide for the Care and Use of LaboratoryAnimals” (NIH publication No. 80-23, revised 1985). After an overnightfast, animals are sedated with ketamine hydrochloride (20 mg/kg).Following the induction of anesthesia with thiopental (12 mg/kg) theanimals are intubated and connected to a ventilator that administers amixture of oxygen and nitrous oxide (1:2 [vol/vol]). Anesthesia ismaintained with 0.5-2.5 vol % isoflurane. Antibiotic prophylaxis isprovided by an intramuscular injection of 1,000 mg of a mixture ofprocaine penicillin-G and benzathine penicillin-G (streptomycin).

Under sterile conditions, an arteriotomy of the left carotid artery isperformed and a 8F-introducer sheath is placed in the left carotidartery. All animals are given 100 IU of heparin per kilogram of bodyweight. Additional 2,500 IU boluses of heparin are administeredperiodically throughout the procedure in order to maintain an activatedclotting time above 300 seconds. A 6F guiding catheter is introducedthrough the carotid sheath and passed to the ostia of the coronaryarteries. Angiography is performed after the administration of 200 ug ofintra coronary nitro glycerin and images analyzed using a quantitativecoronary angiography system. A 3F-embolectomy catheter is inserted intothe proximal portion of the coronary artery and passed distal to thesegment selected for stent implantation and the endothelium is denuded.A coated R stent incorporating an anti-CD34 antibody is inserted throughthe guiding catheter and deployed in the denuded segment of the coronaryartery. Bare stainless steel stents or stents coated with the matrix butwithout antibodies are used as controls. Stents are implanted intoeither the Left Anterior Descending (LAD) coronary artery or the RightCoronary Artery (RCA) or the Circumflex coronary artery (Cx) at a stentto artery ration of 1.1. The sizing and placement of the stents isevaluated angiographically and the introducer sheath was removed and theskin closed in two layers. Animals are placed on 300 mg of ASA for theduration of the experiment.

Animals are sacrificed at 1, 3, 7, 14, and 28 days after stentimplantation. The animals are first sedated and anesthetized asdescribed above. The stented coronary arteries are explanted with 1 cmof non-stented vessel proximal and distal to the stent. The stentedarteries are processed in three ways, histology, immunohistochemistry orby Scanning Electron Microscopy.

For immunohistochemistry the dissected stents are gently flushed with10% Formalin for 30 seconds and the placed in a 10% Formalin/PBSsolution until processing. Stents destined for immunohistochemistry areflushed with 2% Paraformaldehyde (PFA) in PBS for 30 seconds and thenplaced in a 2% PFA solution for 15 min, washed and stored in PBS untilimmunohistochemistry with rabbit anti-human VEGFR-2 or mouse anti-humanTie-2 antibodies is performed.

Stents are prepared for SEM by flushing with 10% buffered Formalin for30 seconds followed by fixation with 2% PFA with 2.5% glutaraldehyde in0.1 M sodium cacodylate buffer overnight. Samples are then washed 3×with cacodylate buffer and left to wash overnight. Post-fixation wascompleted with 1% osmium tetroxide (Sigma) in 0.1M cacodylate bufferwhich is followed by dehydration with ethanol (30% ehtanol, 50%, 70%,85%, 95%, 100%, 100%) and subsequent critical point drying with CO₂.After drying, samples are gold sputtered and visualized under SEM.(Reduction in thrombotic events with heparin-coated Palmaz-Schatz stentsin normal porcine coronary arteries, Circulation 93:423-430,incorporated herein by reference).

For histology the stented segments are flushed with 10% bufferedFormalin for 30 seconds followed by fixation with 10% buffered Formalinuntil processed. Five sections are cut from each stent; 1 mm proximal tothe stent, 1 mm from the proximal end of the stent, mid stent, 1 mm fromthe distal edge of the stent and 1 mm distal to the stent. Sections arestained with Hematoxylin & Eosin (HE) and Elastin Trichrome.

FIGS. 14A-14G show explants taken 1 (FIGS. 14A and 14B) and 48 hours(FIGS. 14C-14G) after implantation and observed under scanning electronmicroscope. The photomicrographs clearly show that the dextran/anti-CD34antibody-coated stents (14B, 14E-G) have capture progenitor endothelialcells as shown by the spindle-shaped appearance of the cells at highermagnification (400×) at 48 hours compared to the dextran-coated control(14A, 14C and 14D).

Cross-sections of the explants from the swine coronary arteries alsoshowed that the dextran-anti-CD34 antibody-coated (14L, 14M) caused apronounced inhibition of intimal hyperplasia (thickness of the arterialsmooth muscle layer) compared to the controls (bare stainless steel 14Hand 14I; dextran-coated 14J and 14K). Fullerene-coated stent implantsalso inhibit intimal hyperplasia better than bare, control stainlesssteel stents as shown in FIGS. 13B-13D.

FIGS. 15A and 15B show, respectively, confocal photomicrographs of 48hours explants of a dextran-plasma coated stent without antibody on issurface, and a dextran-plasma coated anti-CD34 antibody-stent of 18 mmin length. The stents had been implanted into the coronary artery ofjuvenile male Yorkshire swine. The explants were immunohistochemicallyprocessed and stained for VEGFR-2, followed by FITC-conjugated secondaryantibody treatment and studied under confocal microscopy. FIGS. 15B and15C show that the antibody containing stent is covered with endothelialcells as demonstrated by the green fluorescence of the section comparedto the complete lack of endothelium on the stent without antibody (FIG.15A).

Example 6

Incorporation of an Endothelial Growth Factor into Immobilized AntibodyMatrices Applied to Stents: The following describes the steps forimmobilizing an antibody directed toward endothelial progenitor cellscell surface antigens to a biocompatible matrix applied to anintravascular stent to which an endothelial growth factor is thenabsorbed for the enhanced attachment of circulating endothelialprogenitor cells and their maturation to functional endothelium when incontact with blood.

Matrix Deposition: Using methods know to those skilled in the art,stainless steel stents are treated with a plasma deposition to introduceamine functionality on the stent surface. A layer of carboxy functionaldextran (CMDX) will be bound to the amine functional layer deposited onthe stent through the activation of the CMDX carboxyl groups usingstandard procedures, known as water soluble carbodiimide couplingchemistry, under aqueous conditions to which the amine groups on theplasma deposited layer to form an amide bond between the plasma layerand the functional CDMX.

Antibody Immobilization: Antibodies directed toward endothelialprogenitor cells cell surface antigens, e.g., murine monoclonalanti-humanCD34, will be covalently coupled with the CDMX coated stentsby incubation in aqueous water soluble carbodiimide chemistry ina.buffered, acidic solution.

Absorption of Growth Factor: Subsequent to the immobilization of themonoclonal anti-humanCD34 to a CMDX matrix applied to a stent, thedevice is incubated in an aqueous solution of an endothelial growthfactor, e.g. Angiopoietin-2, at an appropriate concentration such thatthe growth factor is absorbed into the CMDX matrix. The treated devicesare rinsed in physiologic buffered saline solution and stored in asodium azide preservative solution.

Using standard angiographic techniques, the above described devices whenimplanted in porcine coronary arteries and exposure to human bloodproduce an enhanced uptake and attachment of circulating endothelialprogenitor cells on to the treated stent surface and accelerate theirmaturation into functional endothelium. The rapid establishment offunctional endothelium is expected to decrease device thrombogenicityand modulate the extent of intimal hyperplasia.

Example 7

Immobilization of an Endothelial Growth Factor and an Antibody on toStents: The following describes the steps for immobilizing an antibodydirected toward endothelial progenitor cells cell surface antigens andan endothelial growth factor to a biocompatible matrix applied to anintravascular stent for the enhanced attachment of circulatingendothelial progenitor cells and their maturation to functionalendothelium when in contact with blood.

Matrix Deposition: Matrix Deposition: Using methods know to thoseskilled in the art, stainless steel stents are treated with a plasmadeposition to introduce amine functionality on the stent surface. Alayer of carboxy functional dextran (CMDX) is bound to the aminefunctional layer deposited on the stent through the activation of theCMDX carboxyl groups using standard procedures, known as water solublecarbodiimide coupling chemistry, under aqueous conditions to which theamine groups on the plasma deposited layer to form an amide bond betweenthe plasma layer and the functional CDMX.

Antibody and Growth Factor Immobilization: Antibodies directed towardendothelial progenitor cells cell surface antigens, e.g. murinemonoclonal anti-human CD34, and an endothelial growth factor, e.g.Angiopoietin-2, is covalently coupled with the CDMX coated stents byincubation at equimolar concentrations in a water soluble carbodiimidesolution under acidic conditions. The treated devices are rinsed inphysiologic buffered saline solution and stored in a sodium azidepreservative solution.

Using standard angiographic techniques, the above described devices whenimplanted in porcine coronary arteries and exposure to human bloodproduce an enhanced uptake and attachment of circulating endothelialprogenitor cells on to the treated stent surface and accelerate theirmaturation into functional endothelium. The rapid establishment offunctional endothelium is expected to decrease device thrombogenicityand modulate the extent of intimal hyperplasia.

Example 8

Small Molecule Functionalization of a Stent: Progenitor endothelialcells were isolated as described in Example 1. The cells were plated infibronectin-coated slides and grown for 7 days in EBM-2 culture medium.Cells were fixed and stained with Propidium Iodine (PI) and aFITC-conjugated endothelial cell specific lectin. (Ulex Europaeus Uea 1)The results of these experiments are shown in FIGS. 16A and 16B. Thefigures show that progenitor endothelial cells are bound to thefibronectin-coated slides and that the cells express a ligand for thelectin on their surface.

Example 9

Transfection of Mammalian Cells in vitro for Use in Blood VesselRemodeling: Progenitor endothelial cells are transfected usingelectroporation of a bicistronic plasmid containing genes encoding aprotein responsible for the production of adenosine and a prostatespecific cell membrane protein. Both genes are under the control oftheir own promoter, so that the genes are expressed constitutively.

1. A therapeutic system for treating a disease in a patient, the systemcomprising: genetically-altered mammalian cells, comprising exogenousnucleic acid encoding a genetically-engineered cell membrane marker andat least one therapeutic gene product; a medical device for implantationinto the patient comprising a coating; said coating comprising a matrixbearing at least one ligand, wherein said ligand recognizes and bindssaid cell membrane marker of said genetically-altered mammalian cells,and wherein said genetically-altered mammalian cells bind to saidmedical device and express and secrete said at least one therapeuticgene product.
 2. The therapeutic system of claim 1, further comprising aactivating molecule for stimulating said genetically-altered mammaliancells to express and/or secrete said therapeutic gene product.
 3. Thetherapeutic system of claim 1, wherein said genetically-alteredmammalian cells are autologous, allogenic, or xenogenic.
 4. Thetherapeutic system of claim 3, wherein said autologous, allogenic, orxenogenic cells are progenitor or mature endothelial cells.
 5. Thetherapeutic system of claim 1, wherein said exogenous nucleic acidpresent in said genetically-altered mammalian cells comprises a DNA oran RNA molecule comprising at least one gene encoding at least onetherapeutic gene product.
 6. The therapeutic system of claim 1, whereinsaid exogenous nucleic acid is extrachromosomal DNA.
 7. The therapeuticsystem of claim 5, wherein said DNA molecule comprises a plasmid.
 8. Thetherapeutic system of claim 6, wherein said extrachromosomal DNAcomprises a regulatory cassette, a cell membrane specific gene and atleast one gene which encodes a peptide for treating a disease.
 9. Thetherapeutic system of claim 8, wherein said cell membrane specific geneencodes an osteogenic or a prostatic cell membrane protein.
 10. Thetherapeutic system of claim 5, wherein said at least one gene encodes atherapeutic gene product selected from the group consisting of vascularendothelial growth factor, angiogenin, anti-angiogenic factor, andfibroblast growth factor.
 11. A method for treating disease in apatient, said method comprises: providing genetically-altered mammaliancells to said patient; comprising exogenous nucleic acid encoding agenetically-engineered cell membrane marker and at least one therapeuticgene product; implanting a medical device comprising a coating into saidpatient; said coating comprising a matrix bearing at least one ligand,wherein said ligand recognizes and binds said cell membrane marker ofsaid genetically-altered mammalian cells, and wherein saidgenetically-altered mammalian cells bind to said medical device andexpress and secrete said at least one therapeutic gene product.
 12. Thesystem of claim 11, further comprising a compound for stimulating thegenetically-altered mammalian cells to express and/or secrete a specificgene product;
 13. The system of claim 11, wherein thegenetically-altered mammalian cells are autologous, allogenic orxenogenic.
 14. A method for the treatment of cancer, comprising:providing genetically-altered mammalian cells to said patient;comprising exogenous nucleic acid encoding a genetically-engineered cellmembrane marker and at least one therapeutic gene product; implanting amedical device comprising a coating into said patient; said coatingcomprising a matrix bearing at least one ligand, wherein said ligandrecognizes and binds said cell membrane marker of saidgenetically-altered mammalian cells, and wherein saidgenetically-altered mammalian cells bind to said medical device andexpress and secrete said at least one therapeutic gene product.
 15. Themethod of claim 14, further comprising the step of stimulating thegenetically-altered mammalian cells to express and/or secrete thetherapeutic gene product.
 16. The method of claim 14, wherein saidgenetically-altered mammalian cells are autologous, allogenic orxenogenic.
 17. The method of claim 15, wherein the step of stimulatingthe genetically-altered mammalian cells to express and/or secrete atherapeutic gene product comprises releasing a compound into thebloodstream, which interacts with the genetically-altered mammaliancells.
 18. The method of claim 16, wherein the autologous, allogenic orxenogenic cells are mature endothelial cells.
 19. The method of claim14, wherein the genetically-altered cells comprise an exogenouslyadministered DNA or RNA molecule comprising at least one gene encodingat least one therapeutic gene product.
 20. The therapeutic system ofclaim 5, wherein said DNA molecule is a plasmid.
 21. The therapeuticsystem of claim 6, wherein said extrachromosomal DNA comprises aregulatory cassette, a cell membrane specific gene and at least one genewhich encodes a peptide for treating a disease.
 22. The therapeuticsystem of claim 8, wherein said cell membrane specific gene encodes anosteogenic or a prostatic cell membrane protein.
 23. The therapeuticsystem of claim 5, wherein said at least one gene encodes a therapeuticgene product selected from the group consisting of vascular endothelialgrowth factor, angiogenin, anti-angiogenic factor, and fibroblast growthfactor.
 24. The method of claim 15, wherein the therapeutic gene productis selected from the group consisting of vascular endothelial growthfactor, angiogenin, anti-angiogenic factor, and fibroblast growthfactor.
 25. A drug delivery system, comprising: genetically-alteredmammalian cells, comprising exogenous nucleic acid encoding agenetically-engineered cell membrane marker and at least one therapeuticgene product; a medical device for implantation into the patientcomprising a coating; said coating comprising a matrix bearing at leastone ligand, wherein said ligand recognizes and binds said cell membranemarker of said genetically-altered mammalian cells, and wherein saidgenetically-altered mammalian cells bind to said medical device andexpress and secrete said at least one therapeutic gene product.
 26. Thedrug delivery system of claim 25, further comprising an activatingmolecule or inducible promoter for stimulating said genetically-alteredmammalian cells to express and/or secrete said therapeutic gene product.27. The therapeutic system of claim 25, wherein said genetically-alteredmammalian cells are autologous, allogenic or xenogenic.
 28. The drugdelivery system of claim 27, wherein said autologous, allogenic orxenogenic cells are mature endothelial cells.
 29. The drug deliverysystem of claim 25, wherein said exogenous nucleic acid present in saidgenetically-altered mammalian cells comprises an exogenous DNA or RNAvector comprising at least one gene encoding at least one therapeuticgene product.
 30. The drug delivery system of claim 25, wherein saidexogenous nucleic acid is extrachromosomal DNA.
 31. The drug deliverysystem of claim 25, wherein said DNA vector comprises a plasmid.
 32. Thedrug delivery system of claim 30, wherein said extrachromosomal DNAcomprises a regulatory cassette, a cell membrane specific gene and atleast one gene which encodes a peptide for treating a disease.
 33. Thedrug delivery system of claim 32, wherein said cell membrane specificgene encodes an osteogenic or a prostatic cell membrane protein.
 34. Thedrug delivery system of claim 29, wherein said at least one gene encodesa therapeutic gene product selected from the group consisting ofvascular endothelial growth factor, angiogenin, anti-angiogenic factor,and fibroblast growth factor.
 35. A method for recruiting cells to ablood contacting surface in vivo, comprising: providing a bloodcontacting surface positioned in the blood stream of a subject, saidblood contacting surface configured to recruit target cells circulatingin the blood stream of said subject to said blood contacting surface;and recruiting said target cells to said blood contacting surface. 36.The method of claim 35, wherein said blood contacting surface comprisesthe luminal surface of a medical device implanted into the subject. 37.The method of claim 35, wherein the step of recruiting said target cellsto the blood contacting surface comprises at least one type of ligandattached to said surface, which ligands recognize and bind said targetcells.
 38. The method of claim 37, wherein the ligands are selected fromthe group cosisting of proteins, lipoproteins, glycoproteins,antibodies, antibody fragments, small molecules, peptides, enzymes,organic catalysts, ribozymes, organometallics, polyamino acids, nucleicacids, sterioidal molecules, antibiotics, antimycotics, cytokines,carbohydrates, oleophobics, lipids and viruses.
 39. The method of claim35, wherein the antibodies or antibody fragments are monoclonalantibodies.
 40. The method of claim 35, wherein the target cells areselected from the group consisting of circulating blood cells,endothelial cells, progenitor endothelial cells, mammalian cells, andgenetically-altered mammalian cells.
 41. The method of claim 35, whereinthe genetically-altered mammalian cells comprise an exogenous DNA or RNAmolecule encoding at least one gene which expresses at least onetherapeutic gene product.
 42. The method of claim 41, wherein thetherapeutic gene product is selected from the group consisting ofvascular endothelial growth factor, angiogenin, anti-angiogenic factor,and fibroblast growth factor.
 43. A method for generating aself-endothelializing medical device in situ, the method comprising: (a)providing a biodegradable scaffolding configured to function as atemporary device; the scaffolding having a luminal surface and anexterior surface; (b) imprlanting the biodegradable scaffolding into ablood vessel; (c) recruiting circulating endothelial progenitor cells tothe luminal surface of the biodegradable scaffolding to form aneo-endothelium; (d) encapsulating the exterior surface of thescaffolding by vascular tissue to form an exterior hemostatic vascularstructure; and (e) degrading the biodegradable scaffolding under in vivoconditions within a time frame which allows the neo-endothelium and theexterior vascular structure to form a functional neo-vessel.
 44. Aprosthesis, comprising: (a) a support member having an exterior surfaceand a blood contacting surface; (b) a first layer of a cross-linkedpolymeric compound coated onto said blood contacting surface of saidsupport member; and, (c) a second layer coated on said first layer, saidsecond layer comprising at least one ligand having an affinity for atarget cell in vivo.
 45. A method for generating a self-endothelializinggraft in vivo, the method comprising: (a) providing a scaffoldingconfigured to function as a vascular graft, said scaffolding having alumen surface and exterior surface, said lumen surface comprisingligands specific for binding to endothelial progenitor cells; (b)implanting said scaffolding into a blood vessel of a subject; and (c)recruiting circulating endothelial progenitor cells to said lumensurface of said scaffolding to form a neo-endothelium.
 46. A method forgenerating a self-endothelializing graft in situ, the method comprising:(a) providing a prosthetic structure having a surface exposed tocirculating blood; (b) implanting the prosthetic structure into asubject; and (c) recruiting circulating progenitor endothelial cellsfrom the blood to the surface of the prosthetic structure to form aneo-endothelium thereon.
 47. A method for generating aself-endothelializing graft in situ, the method comprising: (a)providing a biodegradable scaffolding configured to function as atemporary vascular graft, the scaffolding having a lumen surface andexterior surface; (b) implanting the biodegradable scaffolding into ablood vessel; (c) recruiting circulating progenitor endothelial cells tothe lumen surface of the biodegradable scaffolding to form aneo-endothelium; (d) encapsulating the exterior surface of thescaffolding by vascular tissue to form an exterior hemostatic vascularstructure; and (e) degrading the biodegradable scaffolding under in vivoconditions within a time frame which allows the neo-endothelium and theexterior vascular structure to form a functional neo-vessel.
 48. Abiodegradable scaffolding for forming an endothelialized vascular graftin situ, the scaffolding comprising: (a) a porous biodegradable supportmember having a lumen and an exterior surface; and (b) the lumen surfacecomprising a first layer of at least one species of a polymeric compoundcoated to the support member, and wherein the compound is cross-linkedto itself with a cross-linking agent that forms covalent bonds that aresubject to enzymatic cleavage or non-enzymatic hydrolysis under in vivoconditions.
 49. A method for generating a self-endothelializing graft insitu, the method comprising: (a) providing a prosthetic structure,having a surface exposed to circulating blood; (b) implanting theprosthetic structure into a subject; and (c) recruiting circulatingprogenitor endothelial cells from the blood to the surface of theprosthetic structure to form a neo-endothelium.
 50. The method of claim49, wherein the lumen surface is modified to comprise a ligand specificfor binding the endothelial progenitor cells to the lumen surface of thebiodegradable scaffolding.
 51. A prosthesis for forming anendothelialized vascular graft in situ, the prosthesis comprising: (a) asupport member having a surface; and (b) the surface comprising a firstlayer of at least one species of a polymeric compound coated to thesupport member, and wherein the compound is cross-linked to itself witha cross-linking agent that forms covalent bonds that are subject toenzymatic cleavage or non-enzymatic hydrolysis under in vivo conditions.52. The prosthesis of claim 44, wherein said support member comprises amaterial selected from the group consisting of stainless steel, nitinol,titanium, gold, silicone, superelastic alloys, polytetrafluoroethylene,polyethylene terephthalate, polyesters, and polyethylenes.
 53. Themethod of claim 35 or 44, wherein said target cells comprise autologouscells.
 54. The method of claim 35, wherein said target cells comprisedonor cells.
 55. The method of claim 35, wherein said target cellscomprise cells harvested from bone marrow or fat tissue.
 56. The methodof claim 35, wherein said introducing said target cells comprisesinjecting said target cells into said bloodstream of said subject. 57.The method of claim 35, wherein said coating further comprisesintroducing a layer of polymeric compound onto said blood contactingsurface.
 58. The method of claim 57, wherein said polymeric compound iscross-linked with a cross-linking agent that forms covalent bondscapable of enzymatic cleavage under in vivo conditions.
 59. The methodof claim 57, wherein said polymeric compound is cross-linked with across-linking agent that forms covalent bonds that are capable ofnon-enzymatic hydrolysis under in vivo conditions.
 60. The method ofclaim 59, wherein said cross-linking agent comprises a compound havingat least two reactive functional groups selected from the groupconsisting of aldehydes, epoxides, acyl halides, alkyl halides,isocyanates, amines, anhydrides, acids, alcohols, haloacetals, arylcarbonates, thiols, esters, imides, vinyls, azides, nitros, peroxides,sulfones, maleimides, poly(acrylic acid), vinyl sulfone, succinylchloride, polyanhydrides, succinimidyl succinate-polyethylene glycol,and succinimidyl succinamide-polyethylene glycol, amine reactive esters.61. A kit for recruiting target cells to a blood contacting surfacecomprising: a coating comprising a ligand specific for a circulatingtarget cell, and said coating configured to form a layer on a bloodcontacting surface in vivo.
 62. The kit of claim 61, further comprisingcultured target cells, said target cells comprising a binding partnermolecule for said ligand.
 63. The kit of claim 61, and instructions forusing said kit.
 64. A method for recruitment of cells to a bloodcontacting surface ex vivo, comprising; (a) providing a blood contactingsurface positioned in the blood stream of a subject, said bloodcontacting surface configured to recruit target cells circulating in theblood stream of said subject to said blood contacting surface; and (b)recruiting target cells to said blood contacting surface.
 65. The methodof claim 35, further comprising altering a surface characteristic ofsaid blood contacting surface by said target cells.
 66. The method ofclaim 65, wherein altering of said blood contacting surface by saidtarget cells facilitates the in vivo formation of a cellular tissue onsaid blood contacting surface.
 67. The method of claim 66, wherein saidcellular tissue is a tissue selected from the group consisting ofendothelial, fibrous, epithelial, and bone tissue.
 68. The method ofclaim 35, wherein said ligand is capable of binding to a surfacemolecule of said target cells, said surface molecule selected from thegroup consisting of CD34, CD133, polysaccharides, KDR, P-selectin,glycophorin, CD4, integrins, lectins, and cadherins.
 69. The prosthesisof claim 44, wherein said coating comprises introducing a polymericmaterial onto said blood contacting surface, said polymeric materialcapable of providing controlled release over time of a protein capableof mobilizing said targeted cells.
 70. The method of claim 35, whereinsaid blood contacting surface comprises a coating and said bloodcontacting surface further comprises at least one compound whichpromotes differentiation of said targeted cells on said blood contactingsurface.
 71. The method of claim 70, wherein said blood contactingsurface comprises at least one compound capable of promoting progenitorendothelial cell differentiation.
 72. The method of claim 35, furthercomprising introducing said target cells into said bloodstream of saidsubject.
 73. The method of claim 35, wherein said target cells comprisesinjecting a modifying compound into said bloodstream of said subject.74. The prosthesis of claim 44, wherein said prosthesis is selected fromthe group consisting of a stent, an anastomotic device, a diagnosticdevice, a pacemaker, a heart valve, a vascular graft, a synthetic organ,an artificial heart, a prosthesis, a drug delivering pump, a graft, anautologous graft, a homograft, a xenograft, and a tissue engineeredgraft.